US8088621B2 - Adenoviral fiber exchange shuttle system - Google Patents

Adenoviral fiber exchange shuttle system Download PDF

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US8088621B2
US8088621B2 US11/901,275 US90127507A US8088621B2 US 8088621 B2 US8088621 B2 US 8088621B2 US 90127507 A US90127507 A US 90127507A US 8088621 B2 US8088621 B2 US 8088621B2
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plasmid
selectable marker
cell
lox
shuttle
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Ronald Rodriguez
Shawn Edward Lupold
Wasim Haider Chowdhury
Tarana A. Kudrolli
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Johns Hopkins University
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N2710/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011Details
    • C12N2710/10011Adenoviridae
    • C12N2710/10311Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011Details
    • C12N2710/10011Adenoviridae
    • C12N2710/10311Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345Special targeting system for viral vectors

Definitions

  • adenoviruses are currently used for a variety of purposes, including gene transfer in vitro, vaccination in vivo, and gene therapy.
  • Several features of adenovirus biology have made such viruses the vectors of choice for certain of these applications.
  • adenoviruses transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of virus and high levels of transgene expression can generally be obtained.
  • the genome of the most commonly used human adenovirus (serotype 5) consists of a linear, 36 kb, double-stranded DNA molecule. Both strands are transcribed and nearly all transcripts are heavily spliced.
  • Viral transcription units are conventionally referred to as early (E1, E2, E3 and E4) and late, depending on their temporal expression relative to the onset of viral DNA replication.
  • the high density and complexity of the viral transcription units poses problems for recombinant manipulation, which is therefore usually restricted to specific regions, particularly E1, E2A, E3, and E4.
  • transgenes are introduced in place of E1 or E3, the former supplied exogenously.
  • the E1 deletion renders the viruses defective for replication and incapable of producing infectious viral particles in target cells;
  • the E3 region encodes proteins involved in evading host immunity, and is dispensable for viral production per se.
  • the desired recombinants are identified by screening individual plaques generated in a lawn of packaging cells.
  • the low efficiency of homologous recombination, the need for repeated rounds of plaque purification, and the long times required for completion of the viral production process have hampered more widespread use of adenoviral vector technology.
  • the instant invention provides a methods for making recombinant viral vectors and provides methods and compositions for using these vectors.
  • the instant invention provides methods for generating a recombinant adenoviral vector comprising a desired gene, comprising the steps of co-transforming a cell expressing RecA with a linearized shuttle plasmid comprising a selectable marker, and a transfer plasmid wherein the transfer plasmid comprises a fiber gene, thereby allowing recombination of the plasmids to generate a recombinant adenoviral vector.
  • the transfer plasmid is constructed by co-transforming a donor plasmid and a acceptor plasmid into a cell expressing a Cre recombinase, wherein the acceptor plasmid comprises a nucleic acid segment encoding a negatively selectable marker flanked by lox sites, and a first selectable marker, and the donor plasmid comprises a nucleic acid segment encoding the fiber gene flanked by lox sites and a second selectable marker, thereby allowing for recombination of the fiber gene and the negatively selectable marker.
  • the lox sites are incompatible. In an further related embodiment, the lox sites are mutated to result in unidirectional recombination.
  • the donor plasmid lox sites are Lox m2/66 and Lox 71 and the acceptor plasmid lox sites are Lox m2/71 and Lox 66.
  • the negatively selectable marker is SacB.
  • the fiber gene is modified. In one embodiment, the fiber gene is modified to incorporate a unique restriction site. In an exemplary embodiment, the unique restriction site is in the HI loop. In a further exemplary embodiment, the unique restriction site is a BspEI site.
  • the acceptor plasmid contains a kanamycin selectable marker. In another embodiment, the donor plasmid contains a ampicillin selectable marker.
  • the method further comprises selecting recombinant adenoviral vectors using the selectable marker.
  • the selectable marker is kanamycin.
  • the cell is a bacterial cell, e.g., an E. coli cell. In other embodiments, the cell is a mammalian cell.
  • the shuttle plasmid comprises a resistance gene and a nucleic acid segment encoding a desired product.
  • the desired product is a polypeptide or fragment thereof, a nucleic acid, a siRNA, an RNAi, an shRNA, or an aptamer.
  • the desired product is a polypeptide, e.g., a therapeutic polypeptide.
  • the nucleic acid segment is under control of a promoter.
  • the promoter is a tissue specific promoter.
  • the shuttle plasmid contains a unique restriction site located between RecA recombination sites.
  • the unique restriction site is a Pme I site.
  • this Pme I site can be used to linearize the plasmid.
  • the shuttle plasmid further comprises RecA homologous recombination sites, e.g., Ad5 left and Ad5 right.
  • the transfer plasmid further comprises RecA homologous recombination sites, e.g., Ad5 left and Ad5 right.
  • the instant invention provides methods for generating a recombinant adenoviral vector comprising a desired gene, comprising the steps of co-transforming a cell expressing RecA with a linearized shuttle plasmid comprising a selectable marker and a transfer plasmid wherein the transfer plasmid comprises a fiber gene, wherein the transfer plasmid is constructed by co-transforming a donor plasmid and a acceptor plasmid into a cell expressing Cre recombinase, wherein the acceptor plasmid comprises a nucleic acid segment encoding a negatively selectable marker flanked by lox sites, and a first selectable marker, and the donor plasmid comprises a nucleic acid segment encoding the fiber gene flanked by lox sites and a second selectable marker, thereby allowing for recombination of the fiber gene and the negatively selectable marker, thereby allowing recombination of the plasmids to generate a recombinant
  • the instant invention provides methods of generating a recombinant adenoviral vector comprising a desired gene, comprising the steps of co-transforming a cell expressing the Cre recombinase with a donor plasmid comprising a nucleic acid segment encoding the fiber gene flanked by lox sites and a shuttle-acceptor plasmid comprising a nucleic acid segment encoding a negatively selectable marker flanked by lox sites, and a nucleic acid segment encoding a desired product, thereby allowing recombination of the plasmids to generate a recombinant adenoviral vector.
  • the desired product is a polypeptide, polypeptides, or fragments thereof, a nucleic acid, a siRNA, an RNAi, an shRNA, or an aptamer.
  • the shuttle-acceptor plasmid is constructed by co-transforming a cell expressing RecA with a linearized shuttle plasmid and an acceptor plasmid comprising a negatively selectable marker.
  • the lox sites are incompatible. In a related embodiment, the lox sites are mutated to result in unidirectional recombination.
  • the donor plasmid lox sites are Lox m2/66 and Lox 71 and the acceptor plasmid lox sites are Lox m2/71 and Lox 66.
  • the negatively selectable marker is SacB.
  • the fiber gene is modified.
  • the fiber gene is modified to include a unique restriction site in the HI loop.
  • the restriction site is a BspEI site.
  • the acceptor plasmid contains a kanamycin selectable marker.
  • the donor plasmid contains an ampicillin selectable marker.
  • the methods further comprise selecting recombinant adenoviral vectors using the selectable marker.
  • the selectable marker is kanamycin.
  • the cell is a bacterial cell, e.g., an E. coli cell. In another embodiment, the cell is a mammalian cell.
  • the shuttle plasmid comprises a resistance gene and a nucleic acid segment encoding a desired product.
  • the product is a polypeptide, polypeptides, or fragments thereof, a nucleic acid, an aptamer, a siRNA, RNAi, an shRNA, or an aptamer.
  • the product is a polypeptide.
  • the peptide is a therapeutic polypeptide.
  • nucleic acid segment is under control of a promoter.
  • the promoter is a tissue specific promoter.
  • the resistance gene is not the same as the resistance gene in the acceptor plasmid.
  • the shuttle plasmid contains a unique restriction site located between the RecA homologous recombination sites.
  • the unique restriction site is a Pme I site.
  • the shuttle plasmid is linearized with Pme I.
  • the acceptor plasmid further comprises RecA homologous recombination sites.
  • the RecA homologous recombination sites are Ad5 left and Ad5 right.
  • the shuttle plasmid further comprises RecA homologous recombination sites.
  • the RecA homologous recombination sites are Ad5 left and Ad5 right.
  • the invention also provides shuttle plasmids.
  • the instant invention also provides acceptor plasmids.
  • the instant invention provides, shuttle-acceptor plasmids.
  • the instant invention provides donor plasmids.
  • the instant invention provides transfer plasmids.
  • the instant invention provides recombinant viral vectors comprising a resistance gene located between RecA homologous recombination sites, and a nucleic acid segment encoding a desired product.
  • the invention provides a viral vector consisting of the nucleic acid molecule set forth as SEQ ID NO: 1.
  • the invention provides a shuttle plasmid consisting of the nucleic acid sequence set forth as SEQ ID NO:2.
  • the invention provides a donor plasmid consisting of the nucleic acid sequence set forth as SEQ ID NO:3.
  • the invention provides a donor plasmid consisting of the nucleic acid sequence set forth as SEQ ID NO:4.
  • the invention provides a donor plasmid consisting of the nucleic acid sequence set forth as SEQ ID NO: 5.
  • the invention provides an acceptor plasmid consisting of the nucleic acid sequence set forth as SEQ ID NO:6.
  • the instant invention provides methods of creating virus comprising linearizing the viral vectors described herein and transfecting the linearized vector into a cell, thereby creating a virus.
  • the invention also provides methods of making psuedotyped virus using the viral vectors described herein.
  • the invention provides methods of treating an individual in need of treatment by administering to the individual a viral vector or a virus described herein.
  • FIG. 1 is a schematic representation of a lox site showing two inverted repeats separated by a spacer region (SEQ ID NO.9).
  • FIGS. 2A-B depict various lox sequences.
  • FIG. 2A depicts lox sequences with half-site mutations in italics.
  • FIG. 2B depicts spacer sequences with mutations in italics (SEQ ID NOS.9 and 11-13).
  • FIG. 3 is a schematic depicting two non-compatible spacer sequences (black arrows) that force gene exchange rather than excision.
  • the reaction of two half-mutant lox sites results in a dually mutated lox site (PR-SacB) and a unidirectional reaction (SEQ ID NOS.12-15).
  • FIG. 4 is a schematic of pFex fiber exchange followed by RecA recombination resulting in pShuffle-Fib, an adenoviral vector.
  • This vector can be digested with Pac I and transfected into a desired cell line to create virus.
  • FIG. 5 is a schematic of Rec A recombination followed by pFex fiber exchange.
  • the pshuttle-Fib is the completed adenoviral vector. This vector can be digested with Pac I and transfected into a desired cell line to create virus.
  • FIG. 6 is a schematic depicting step 1 of pFex assembly.
  • FIG. 7 is a schematic depicting step 2 of pFex assembly.
  • FIG. 8 is a schematic depicting step 3 of pFex assembly.
  • FIG. 9 is a schematic showing vectors pFex and pFex-p*.
  • FIG. 10 is a schematic depicting Fiber Shuttle Lox m2/71.
  • FIG. 11 is a schematic of RP-Fib-1.
  • FIG. 12 is a schematic of RPuc-Fib-1.
  • FIG. 13 is a schematic of pAdTrack shuttle vector.
  • FIG. 14 is a schematic of pAdTrack-CMV-Luc shuttle vector.
  • FIG. 15 depicts the results of restriction digests demonstrating pFex recombination with pAdTrack vectors.
  • FIG. 16 depicts the results of restriction digests demonstrating ColE1/Ad Right hand recombination.
  • FIG. 17 depicts the results of restriction digests indicating the expected products by co-transformation of fiber shuttle and pFex into 294cre cells or by transformation of fiber shuttle into pFex stable 294cre cells (b294-fex).
  • FIG. 18 depicts the results of restriction digests indicating that transformants contained the desired products.
  • FIG. 19 depicts the results of restriction digests indicating that all products have the expected molecular weight.
  • Track-Fib refers to pAdTrack recombinants and Luc-Fib refers to the pAdTrack-Luc-Fib recombinants.
  • FIG. 20 is a schematic of Rpuc-WTFib, a fiber shuttle that contains wild-type fiber cDNA.
  • FIG. 21 depicts a comparison of the plaque size of AdTrack-AdEasy and AdTrack-WTFib by fluorescent microscopy.
  • FIG. 22 depicts CRE mediated fiber exchange into viral genome in mammalian 293-cre cells through the use of a pseudotyped pAdTrack-pFex virus.
  • FIG. 23 depicts detargeted AdTrack-Fib2 virus which was generated by pseudotyped AdTrack-Fex recombination with Rpuc-Fib2 in Cre recombinase expressing mammalian cells.
  • FIG. 24 is Table 1 entitled Primers for Constructing and Sequencing pFex (SEQ ID NOS.19-48).
  • FIG. 25 is Table 2 entitled Primers for Constructing and Sequencing Fiber Shuttles (SEQ ID NOS.49-52).
  • FIG. 26 is Table 3 entitled Fiber Shuttle Vectors.
  • FIG. 27 is Table 4 entitled Total Transformants from 294 co-transfections.
  • FIG. 28 is Table 5 entitled Percent Recombinants of Large pAdTrack-Fex Vector.
  • FIG. 29 is Table 6 entitled pFex and AdEasy based virus.
  • FIG. 30 depicts the nucleic acid sequence of pShuttle-Fib (SEQ ID NO: 1).
  • FIG. 31 depicts the nucleic acid sequence of pShuttle (SEQ ID NO:2).
  • FIG. 32 depicts the nucleic acid sequence of RP-Fib (SEQ ID NO:3).
  • FIG. 33 depicts the nucleic acid sequence of RPuc-Fib (SEQ ID NO:4).
  • FIG. 34 depicts the nucleic acid sequence of RP-Blast-Fib (SEQ ID NO: 5).
  • FIG. 35 depicts the nucleic acid sequence of pFEX (SEQ ID NO:6).
  • the instant invention is based, at least in part, on the discovery of methods for generating adenoviral vectors which are more efficient and more flexible than current systems for producing recombinant viral vectors. These methods and vectors are compatible with current technology as described in U.S. Pat. No. 5,922,576, the contents of which is expressly incorporated herein by reference.
  • the instant invention provides a two-stage recombination system for making recombinant adenoviral vectors that has several advantages in terms of ease, sensitivity and flexibility.
  • the instant invention provides methods of producing recombinant adenoviral vectors that offer the practitioner the flexibility to adapt the system to their particular needs. They skilled artisan may introduce the desired product into an already completed transfer vector and select for a recombinant adenoviral vector, or introduce the desired product into the acceptor vector as the initial step in the method, thereby allowing them the flexibility that other systems do not provide. Moreover, the ability to recover very small numbers of recombinant viral particles amongst many transformants is particularly advantageous.
  • the early adenovirus gene products E1A and E1B are deleted and provided in trans by the packaging cell line 293 developed by Frank Graham (Graham et al. (1987) J. Gen. Birol. 36:59-72 and Graham (1977) J. Genetic Virology 68:937-940).
  • the gene to be transduced is commonly inserted into adenovirus in the deleted E1A and E1B region of the virus genome Bett et al. (1994), supra.
  • Adenovirus vectors as vehicles for efficient transduction of genes have been described by Stratford-Perricaudet (1990) Human Gene Therapy 1:2-256; Rosenfeld (1991) Science 252:431-434; Wang et al. (1991) Adv. Exp. Med. Biol.
  • the present invention utilizes recombination, e.g., recombination in bacteria, to combine plasmid DNA molecules containing a desired product to form an adenoviral vector.
  • the instant invention provides methods for generating recombinant adenoviral vectors that utilizes RecA and Cre mediated homologous recombination.
  • Recombination is a process in which two DNA molecules become joined and nucleic acid is exchanged. Homologous recombination occurs between two sequences having regions of homology. Bacterial recombination is particularly robust.
  • identical sequences must be present in both. Using standard methods in the art, segments of the adenoviral genome can be put in the plasmids to create regions of homology.
  • adenovirus vector or “adenoviral vector” (used interchangeably) is a term well understood in the art and generally comprises a polynucleotide comprising all or a portion of an adenovirus genome.
  • adenovirus refers to the virus itself or derivatives thereof. The term covers all serotypes and subtypes and both naturally occurring and recombinant forms, except where otherwise indicated.
  • An adenoviral vector of the present invention can be in any of several forms, including, but not limited to, naked DNA; an adenoviral vector encapsulated in an adenovirus coat; packaged in another viral or viral-like form (such as herpes simplex virus and AAV); encapsulated in a liposome; complexed with polylysine or other biocompatible polymer; complexed with synthetic polycationic molecules; conjugated with transferrin; complexed with compounds such as PEG to immunologically “mask” the molecule and/or increase half-life, or conjugated to a non-viral protein.
  • An adenoviral vector of this invention may be in the form of any of the delivery vehicles described herein. Such vectors are one embodiment of the invention.
  • the polynucleotide is DNA.
  • DNA includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides.
  • adenovirus vectors are replication-competent in a target cell.
  • plasmid denotes an extrachromosomal circular DNA capable of autonomous replication in a given cell.
  • the range of suitable plasmids is very large.
  • the plasmid is designed for amplification in bacteria and for expression in an eukaryotic target cell.
  • plasmids can be purchased from a variety of manufacturers.
  • Exemplary plasmids include but are not limited to those derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), pREP4, pCEP4 (Invitrogene), pCI (Promega) and p Poly (Lathe et al., Gene 57 (1987), 193-201).
  • Plasmids can also be engineered by standard molecular biology techniques (Sambrook et al., Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), N.Y.). It may also comprise a selection gene in order to select or to identify the transfected cells (e.g., by complementation of a cell auxotrophy or by antibiotic resistance), stabilizing elements (e.g., cer sequence; Summers and Sherrat, 1984, Cell 36, 1097-1103) or integrative elements (e.g., LTR viral sequences and transposons).
  • stabilizing elements e.g., cer sequence; Summers and Sherrat, 1984, Cell 36, 1097-1103
  • integrative elements e.g., LTR viral sequences and transposons
  • the term “shuttle plasmid” is intended to mean a plasmid comprising a unique restriction site between RecA homologous recombination sites and used to insert a desired nucleic acid molecule, i.e., a nucleic acid molecule encoding a desired product, into a recombinant adenoviral vector.
  • the RecA homologous recombination sites can be, for example, Ad5 right and Ad5 left.
  • the shuttle plasmid may have a tissue specific promoter which controls the expression of the desired nucleic acid molecule.
  • the shuttle plasmid also contains a majority of the viral genes necessary to form viral particles. However, the shuttle plasmid does not contain all necessary genes to form viral particles.
  • An exemplary shuttle plasmid is referred to as pShuttle herein.
  • RecA mediated homologous recombination is used to exemplify enzyme mediated homologous recombination.
  • Other enzymes capable of mediating homolgous recombination are known in the art and can be used to design the vectors of the invention, and can further be used in the methods of the invention.
  • homologous recombination enzymes are known in eukaryotes, e.g., Rad51, Rad57, Rad55 and DMC1, in Archaea, e.g., RadA and RadB, and in phage, e.g., vsX in phage T4. These enzymes and homologs and orthologs of these enzymes are envisioned for use in the methods of the present invention.
  • transfer plasmid is intended to mean the plasmid that results from the Cre mediated recombination of the donor plasmid and the acceptor plasmid.
  • the transfer plasmid has the fiber gene, or other gene in the fiber location, inserted in place of the negatively selectable marker.
  • the transfer plasmid has RecA homologous recombination sites to allow for insertion of a desired nucleic acid molecule by RecA mediated homologous recombination with the shuttle plasmid.
  • the transfer plasmid also has a selectable marker, i.e., ampicillin located between the RecA homologous recombination sites.
  • the RecA homologous recombination sites can be, for example, Ad5 right and Ad5 left.
  • An exemplary transfer plasmid is referred to as pFex-Fib herein.
  • nucleic acid molecule encoding fiber is intended to mean a nucleic acid segment encoding viral capsid protein that is responsible for mediating high-affinity attachment of adenovirus to a target cell.
  • the amino acid sequence of fiber is available as GenBank Accession number P03275, and is further described by Herisse, J., et al. (1981) Nucleic Acids Res. 9:4023-4042.
  • the fiber gene used in the methods and compositions of the invention can be a functional fragment of the fiber protein, i.e., a fragment that retains the ability to allow the attachment of a virus to a cell.
  • the term “donor plasmid” is intended to mean a plasmid containing a donor gene flanked on either side by lox sites.
  • the donor gene is a fiber gene, or fragment thereof.
  • other genes can be used in place of fiber.
  • another gene that encodes a cell surface recognition protein can be used in place of fiber.
  • a nucleic acid molecule encoding a toxin can be used in place of fiber.
  • the donor plasmid has a different selectable marker than the acceptor plasmid.
  • the donor plasmid has ampicillin, kanamycin, or blastocidin resistance.
  • Exemplary donor plasmids are referred to as RP-Fib, RPuc-Fib, and Rblast-Fib herein.
  • acceptor plasmid is intended to mean a plasmid containing a negatively selectable marker flanked by lox sites and a selectable marker, e.g., ampicillin, located between RecA homologous recombination sites.
  • the negatively selectable marker can be, for example, SacB.
  • An exemplary acceptor plasmid is referred to as pFex herein.
  • shuttle-acceptor plasmid is intended to mean the recombination product of RecA mediated recombination of a shuttle plasmid and an acceptor plasmid.
  • the shuttle-acceptor plasmids of the invention comprise a negatively selectable marker located between two lox sites, a resistance marker, and a nucleic acid molecule encoding a desired product.
  • An exemplary shuttle-acceptor plasmid is referred to as pShuttle-Fex herein.
  • the “desired product” in use in the present invention encodes a gene product of therapeutic interest.
  • a “desired product” can have a therapeutic or protective activity when administered appropriately to a patient, especially a patient suffering from a disease or illness condition or who should be protected against this disease or condition. Such a therapeutic or protective activity can be correlated to a beneficial effect on the course of a symptom of said disease or said condition. It is within the reach of the man skilled in the art to select a gene encoding an appropriate gene product of therapeutic interest, depending on the disease or condition to be treated. In a general manner, his choice may be based on the results previously obtained, so that he can reasonably expect, without undue experimentation, i.e., other than practicing the invention as claimed, to obtain such therapeutic properties.
  • the desired product can be homologous or heterologous to the host cell into which it is introduced.
  • it encodes a polypeptide, a ribozyme or anti-sense RNA, RNAi, an aptamer or the like.
  • polypeptide is to be understood as any translational product of a polynucleotide whatever its size is, and includes polypeptides having as few as 7 residues (peptides), but more typically proteins.
  • it may be from any origin (prokaryotes, lower or higher eukaryotes, plant, virus etc). It may be a native polypeptide, a variant, a chimeric polypeptide having no counterpart in nature or fragments thereof.
  • the gene of interest in use in the present invention encodes at least one polypeptide that can compensate for one or more defective or deficient cellular proteins in an animal or a human organism, or that acts through toxic effects to limit or remove harmful cells from the body.
  • a suitable polypeptide may also be immunity conferring and acts as an antigen to provoke a humoral or a cellular response, or both.
  • the regulatory elements controlling the expression of the desired gene may further comprise additional elements, such as exon/intron sequences, targeting sequences, transport sequences, secretion signal sequences, nuclear localization signal sequences, IRES, polyA transcription termination sequences, tripartite leader sequences, sequences involved in replication or integration. These elements have been reported in the literature and can be readily obtained by those skilled in the art.
  • lox sites is intended to mean a nucleic acid sequence that the Cre recombinase recognizes.
  • the canonical lox site is the loxP site.
  • Lox sites are 34 nucleotides in length and have a 13 base pair inverted repeat separated by an 8 base pair spacer (see FIG. 1 ). Wild-type lox sites are unaltered following recombination thereby allowing for a reversible reaction.
  • the instant invention uses “incompatible” lox sites which have a mutation such that intrageneic recombination, i.e. recombination within a plasmid which can result in deletion or inversion of flanked nucleic acid, can not occur.
  • Exemplary mutations include those to the spacer that result in non-functional lox sites following recombination (see FIGS. 2A-B ).
  • the instant invention also applies “half-mutant” lox sites, which when correctly recombined, produce one fully mutant lox site and one wild type lox site, resulting in a non-functional lox site, thus preventing the reverse reaction.
  • Specific exemplary incompatible lox sites for uni-directional insertion include the Lox m2/66 and Lox 71 on the donor fragment and Lox m2/71 with Lox66 on the acceptor fragment (see, for example, Langer, S. J. et al. (2002) Nucleic Acid Research 20:3067-77)
  • polynucleotide and “nucleic acid”, used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases.
  • polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches.
  • sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.
  • the polynucleotide is DNA.
  • DNA includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides.
  • a key step in the generation of adenoviral plasmids according to the present invention is the co-transformation of bacteria with precursor DNA vectors. Transformation is the introduction of DNA into a bacterial cell. Transformation can be carried out by a number of techniques known in the art. Such methods include but are not limited to electroporation (exposure of a cell suspension to an electrical field), the use of calcium phosphate solutions, and the use of lipids to package the DNA and fuse with the cell membrane. Co-transformation refers to the introduction of two different species of DNA molecule into the same cell.
  • the plasmid desirably comprises one or more desired product.
  • segments of DNA consisting of adenoviral sequences flank the desired product to promote homologous recombination with other nucleic acid molecules to ultimately produce an adenoviral vector.
  • the adenoviral vector typically contains most of the adenoviral genome.
  • the adenoviral vector may also contain a bacterial origin of replication. Portions of the wild-type adenoviral genome may be deleted to permit insertion of desired products and the packaging of recombinant adenoviral vectors containing the desired genes.
  • the invention provides alternative methods for producing recombinant adenoviral vectors.
  • the methods rely on two homologous recombination steps, one mediated by Cre and the other mediated by RecA.
  • the instant invention provides methods in which the Cre mediated recombination must precede the RecA recombination, methods in which the RecA mediated recombination must precede the Cre mediated recombination, and finally methods in which the order of recombination events in immaterial.
  • the order of recombination events is dictated by the resistance genes on the precursor plasmids.
  • the Cre mediated recombination must be preformed first (see, for example, the schematic set forth in FIG. 4 ).
  • the RecA mediated recombination must occur first (see, for example, the schematic set forth in FIG. 5 ).
  • the order of recombination steps is at the discretion of the skilled artisan (for example, if the donor plasmid had blasticidin resistance as described in the examples).
  • a Cre expressing cell is transformed with a donor and acceptor plasmid such that Cre mediated recombination results in the formation of a transfer plasmid.
  • the donor plasmid contains a fiber gene, or other gene product to target the recombinant virus to a specific cell, flanked by lox sites.
  • the acceptor plasmid has a negatively selectable marker, such as SacB, flanked by lox sites.
  • the lox sites are engineered, i.e., mutated, to result in irreversible, uni-directional recombination and to prevent intragenic recombination.
  • Cells containing the recombinant transfer plasmid are selected by growth in media containing a substrate for the negatively selectable marker and an antibiotic for which the resulting transfer plasmid carries a resistance gene.
  • the negatively selectable gene is SacB and the antibiotic resistance is to ampicillin
  • cells containing the recombinant transfer plasmid are selected by growth in media containing sucrose and ampicillin.
  • the shuttle plasmid is linearized using a restriction enzyme that has a single restriction site in the plasmid.
  • shuttle plasmids may not be linearized prior to introducing them into a cell for recombination.
  • Recombinant adenoviral vectors formed as a result of RecA mediated recombination are selected by growing cells in the presence of an antibiotic which the recombinant adenoviral vectors carry a resistance gene against. This resistance gene was originally contained on the shuttle plasmid and is integrated into the recombinant viral vector during RecA mediated recombination.
  • a schematic of this embodiment is set forth in FIG. 4 .
  • the recombinant viral vectors are produced by transforming a cell expressing RecA with a linear shuttle plasmid and an acceptor plasmid.
  • Cells containing a shuttle-acceptor plasmid are selected in media containing an antibiotic to which the resulting shuttle-acceptor plasmid confers resistance.
  • Recombinant shuttle-acceptor plasmids are isolated and transformed into a cell expressing Cre along with a donor plasmid.
  • Recombinant adenoviral vectors are selected using by growing cells in media containing a substrate for the negatively selectable marker and an antibiotic which recombinant adenoviral vectors carry a resistance gene against. This resistance gene was originally contained on the donor plasmid and is integrated into the recombinant viral vector during Cre mediated recombination.
  • a schematic of this embodiment is set forth in FIG. 5 .
  • the Cre-recombinase mediate exchange is not limited to bacteria or plasmids.
  • fiberless acceptor plasmids can be packaged into working virus through complementary cell lines that express fiber protein (a process known as psuedotyping). These pseudotyped acceptor plasmids can then be used to infect Cre expressing cells, e.g., mammalian cells such as 293cre57, that have been transfected with fiber exchange vectors, i.e. donor vectors. Cell lysate and supernatant are then harvested and used to infect a non-Cre expressing packaging line, immediately generating a recombinant adenovirus.
  • Adenoviral particles can be prepared according to any conventional technique in the field of the art, such as homologous recombination in a permissive cell line (e.g., as described in Graham and Prevect, 1991, Methods in Molecular Biology, Vol 7, Gene Transfer and Expression Protocols; Ed E. J. Murray, The Human Press Inc, Clinton, N.J.) or in Escherichia coli (as described in WO96/17070). Propagation is advantageously performed in a complementing cell line or in the presence of a helper virus providing complementation in trans. “Complementing” or “complementation” denotes that the capability to encode and/or express functions that are defective in the vector but necessary for generating viable viral particles.
  • the cell lines 293 (Graham et al., 1977, J. Gen. Virol. 36, 59-72) and PERC6 (Fallaux et al., 1998, Human Gene Therapy 9, 1909-1917) are commonly used to complement the E1 function.
  • Other cell lines have been engineered to complement doubly defective vectors (Yeh et al., 1996, J. Virol. 70, 559-565; Krougliak and Graham, 1995, Human Gene Ther. 6, 1575-1586; Wang et al., 1995, Gene Ther. 2, 775-783; Lusky et al., 1998, J. Virol. 72, 2022-2033; EP919627 and WO97/04119).
  • the adenoviral particles can be recovered from the culture supernatant but also from the cells after lysis and optionally further purified according to standard techniques (e.g., chromatography, ultracentrifugation, as described in WO96/27677, WO98/00524 and WO98/26048). Furthermore, the virions may be amplified by successive passage in a permissive cell in order to generate a high titer viral stock that may be used in the preparation of clinical lots.
  • the recombinant adenovirus vector generated as described above may be used to transfect mammalian cells. Techniques for transfection are well known. Available techniques include but are not limited to electroporation, the use of calcium chloride, and packaging of the vector together with lipid for fusion with the cells of interest. Cells may be transfected with the vector either in vitro or in vivo. The design of the recombinant adenoviral vector may place specific constraints on cells to be transfected. If production of viral particles is desired, a special packaging cell must be used that produces the adenoviral gene products which the adenoviral vector lacks. Which packaging cells are employed to replicate the virus will depend on the composition of the adenoviral vector used.
  • the adenoviral vector may have specific portions of the adenoviral genome deleted, in order to make room for the desired gene in the recombinant vector. Suitable deletions which may be used include those of all or part of adenoviral transcription units E1, E3, and E4.
  • the packaging cells preferably stably express the adenoviral proteins coded by the deleted transcription units. Techniques are known in the art for stably transfecting a cell line with whichever adenoviral sequences are required, i.e., by incorporation of the genes into the cell's genome. If virus particle production is not required, then packaging cell lines need not be used. For example, if cells are to express the desired product, production of viral particles need not be achieved. Thus for in vivo gene therapy, the recipient cells need not be able to complement the defective viruses.
  • Genes encoding a detectable marker may be present in adenoviral vector to allow for detection of the recombinant vector once produced.
  • a marker is used which is easy to monitor. More preferably a marker is used which can be detected even when present at very low levels.
  • Use of a detectable marker permits monitoring of the transfection process.
  • the detectable marker is ⁇ -galactosidase or green fluorescent protein (GFP). Detection of GFP can be achieved, for example, by fluorescence microscopy of cultured cells.
  • Genes encoding a selectable product can also be used as linked markers to the desired product.
  • a selectable product is necessary for growth under a particular set of conditions. Thus it can be used to selectively grow only those cells that have been transformed or transfected.
  • a preferred selectable product is an antibiotic resistance enzyme, such as those for ampicillin, kanamycin, or blastocidin.
  • the adenoviral vector of the invention can also be used to produce a pseudotyped viral particle, i.e., a viral particle that contains one or more structural genes that are not derived from the adenoviral genome.
  • a pseudotyped viral particle i.e., a viral particle that contains one or more structural genes that are not derived from the adenoviral genome.
  • the viral vectors described herein can be made by recombination in intact viral genomes thereby producing pseudotyped virus.
  • Cell type-specific targeting may be achieved with vectors derived from viruses having a broad host range by the modification of viral surface proteins.
  • the specificity of infection of adenoviruses is determined by the attachment to cellular receptors present at the surface of permissive cells.
  • the fiber gene is exemplified throughout the instant application.
  • penton plays a critical role in cellular attachment (Defer et al. J. Virol. 64 (1990) 3661-3673).
  • cell targeting of adenoviruses can be carried out by genetic modification of a viral gene, e.g., fiber and/or penton, to generate modified proteins capable of specific interaction with unique cell surface polypeptides.
  • a viral gene e.g., fiber and/or penton
  • modifications are described in literature (for example in Wickam et al., 1997, J. Virol. 71, 8221-8229; Arnberg et al., 1997, Virol. 227, 239-244; Michael et al., 1995, Gene Therapy 2, 660-668; WO94/10323).
  • a exemplary penton mutant is described herein and called pFex-p* (mutation D342E).
  • the present invention also provides a host cell comprising an adenoviral vector of the invention, a polynucleotide or an expression vector as defined in connection with the use of the invention or infected by a viral particle of the invention.
  • the vector may be inserted into the cellular genome or not (episome).
  • a host cell may be unique type of cells or a group of different types of cells and encompass cultured cell lines, primary cells and proliferative cells, with a special preference for cells of human origin.
  • compositions e.g., pharmaceutical compositions, comprising as an agent an adenoviral vector according to the invention, a polynucleotide or an expression vector as described in connection with the use of the invention, a host cell or a viral particle according to the invention or prepared according to the method of the invention.
  • composition according to the invention may be manufactured in a conventional manner for a variety of modes of administration including systemic, topical and local administration.
  • systemic administration injection is preferred, e.g., intravenous, intraperitoneal, intragastric, subcutaneous, intracardiac, intraarterial, intracoronary, intravascular, intraarterial, intramuscular, intrathecal, intratumoral, intranasal, intrapulmonary or intratracheal routes.
  • Local administration include aerosolization instillation and oral routes of administration.
  • the administration may take place in a single dose or a dose repeated one or several times after a certain time interval.
  • a composition based on viral particles may be formulated in the form of doses of between 10 4 and 10 14 iu (infectious unit), advantageously between 10 5 and 10 13 iu and preferably between 10 6 and 10 12 iu.
  • the titer may be determined by conventional techniques.
  • the doses of DNA vector are preferably comprised between 0.01 and 10 mg/kg, and more especially between 0.5 and 2 mg/kg.
  • the composition of the invention can be in various forms, e.g., solid (powder, lyophilized form) or liquid (e.g., aqueous).
  • the composition comprises a pharmaceutically acceptable carrier, allowing its use in a method for the therapeutic treatment of humans or animals.
  • the carrier is preferably a pharmaceutically suitable injectable carrier or diluent which is non-toxic to a human or animal organism at the dosage and concentration employed (for examples, see Remington's Pharmaceutical Sciences, 16.sup.th ed. 1980, Mack Publishing Co). It is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength, such as provided by a sucrose solution.
  • aqueous or partly aqueous liquid carriers comprising sterile, pyrogen-free water, dispersion media, coatings, and equivalents, or diluents (e.g., Tris-HCl, acetate, phosphate), emulsifiers, solubilizers, excipients or adjuvants.
  • diluents e.g., Tris-HCl, acetate, phosphate
  • emulsifiers e.g., solubilizers, excipients or adjuvants.
  • the pH of the composition is suitably adjusted and buffered in order to be appropriate for use in humans or animals.
  • carriers or diluents for an injectable composition include water, isotonic saline solutions which are preferably buffered at a physiological pH (such as phosphate buffered saline, Tris buffered saline, mannitol, dextrose, glycerol containing or not polypeptides or proteins such as human serum albumin).
  • a physiological pH such as phosphate buffered saline, Tris buffered saline, mannitol, dextrose, glycerol containing or not polypeptides or proteins such as human serum albumin.
  • such a composition may comprise 10 mg/ml mannitol, 1 mg/ml HSA, 20 mM Tris pH 7.2 and 150 mM NaCl.
  • composition according to the present invention may include one or more stabilizing substance(s), such as lipids (e.g., cationic lipids, liposomes, lipids as described in WO98/44143; Felgner et al., 1987, Proc. West. Pharmacol. Soc.
  • lipids e.g., cationic lipids, liposomes, lipids as described in WO98/44143; Felgner et al., 1987, Proc. West. Pharmacol. Soc.
  • nuclease inhibitors 32, 115-121; Hodgson and Solaiman, 1996, Nature Biotechnology 14, 339-342; Remy et al., 1994, Bioconjugate Chemistry 5, 647-654
  • nuclease inhibitors such as WO98/53853
  • collagenase such as collagenase, polymers, chelating agents (EP890362), in order to preserve its degradation within the animal/human body and/or improve delivery into the host cell.
  • Such substances may be used alone or in combination (e.g., cationic and neutral lipids).
  • adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells.
  • the mixture of adenoviruses to solutions containing a lipid-complexed plasmid vector or the binding of DNA to polylysine covalently attached to adenoviruses using protein cross-linking agents may substantially improve the uptake and expression of the vector (Curiel et al., 1992, Am. J. Respir. Cell. Mol. Biol. 6, 247-252).
  • the present invention also provides the use of an adenoviral vector according to the invention, a polynucleotide or an expression vector, as described in connection with the use according to the invention, a viral particle or a host cell according to the invention for the preparation of a medicament intended for gene transfer, preferably into a human or animal body.
  • gene transfer has to be understood as a method for introducing any gene of interest into a cell.
  • immunotherapy that relates to the introduction of a potentially antigenic epitope into a cell to induce an immune response which can be cellular or humoral or both.
  • the adenoviral vector, the polynucleotide and expression vector or the viral particle of the present invention may be delivered in vivo to the human or animal organism by specific delivery means adapted to the pathology to be treated.
  • a balloon catheter or a stent coated with the adenoviral vector, the expression vector carrying the polynucleotide or the viral particle may be employed to efficiently reach the cardiovascular system (as described in Riessen et al., 1993, Hum Gene Ther. 4, 749-758; Feldman and Steg, 1996, Medecine/Science 12, 47-55). It is also possible to deliver said therapeutic agents by direct administration, e.g., intravenously, in an accessible tumor, in the lungs by aerosolization and the like.
  • eukaryotic host cells that have been engineered ex vivo to contain the adenoviral vector, the expression vector carrying the polynucleotide or the viral particle according to the invention.
  • Methods for introducing such elements into an eukaryotic cell include microinjection of minute amounts of DNA into the nucleus of a cell (Capechi et al., 1980, Cell 22, 479-488), transfection with CaPO.sub.4 (Chen and Okayama, 1987, Mol. Cell. Biol. 7, 2745-2752), electroporation (Chu et al., 1987, Nucleic Acid Res.
  • the present invention also relates to a method for the treatment of a human or animal organism, comprising administering to said organism a therapeutically effective amount of an adenoviral vector of the invention, the polynucleotide or expression vector as described in connection with the use according to the invention, a viral particle or an eukaryotic cell according to the invention.
  • a “therapeutically effective amount” is a dose sufficient for the alleviation of one or more symptoms normally associated with the disease or condition desired to be treated. When prophylactic use is concerned, this term means a dose sufficient to prevent or to delay the establishment of a disease or condition.
  • the method of the present invention can be used for preventive purposes and for therapeutic applications relative to the diseases or conditions listed above.
  • the present method is particularly useful to prevent or reduce the establishment of an inflammatory response following administration of a conventional gene-therapy vector. It is to be understood that the present method can be carried out by any of a variety of approaches.
  • the vector, viral particle, cell or the pharmaceutical composition of the invention can be administered directly in vivo by any conventional and physiologically acceptable administration route, for example by intravenous injection, by direct injection into an accessible tumor or by means of an appropriate catheter into the vascular system, etc.
  • the ex vivo approach may also be adopted which consists of introducing the adenoviral vector, the polynucleotide or the viral particle according to the invention into cells, growing the transfected/infected cells in vitro and then reintroducing them into the patient to be treated.
  • a kit according to the invention comprises one or more of the described plasmids, e.g., a shuttle plasmid, a transfer plasmid, a donor plasmid, and/or an acceptor plasmid, useful in the generation of recombinant adenoviral vectors.
  • a user of the kit may insert one or more desired genes into the shuttle plasmid using, for example, a restriction endonuclease and a DNA ligase.
  • the kit may also comprise a packaging cell line for producing virus particles from the defective adenoviral vector and/or the recombinant adenoviral vectors produced containing the desired product.
  • the kit may also comprise bacterial cells which can be used for co-transformation.
  • the bacterial cells are homologous-recombination proficient and highly competent to receive transforming DNA.
  • each kit component is separately packaged to avoid premature mixing. Further, all individually packaged components are provided in a box or other container which holds the other components. Instructions for making a recombinant adenovirus vector according to the methods disclosed herein may also be included in the kit. Reference to instructions may also be provided in the kit, for example to a text or webpage.
  • Kits may also contain the recombinant adenoviral vectors, or viral particles, produced by the methods of the invention and instructions for the administration of the vectors or viral particles to a subject for therapeutic or preventative purposes.
  • the Cre recombinase from bacteriophage P1 is an enzyme which mediates the excision and integration of DNA based on specific sequence binding sites (lox) through stepwise cleavage and ligation involving Holiday Junction intermediates (Ghosh, K. and Van Duyne, G. D. (2002) Methods, 28: 374-383). Though nearly 100 related tyrosine recombinases have been identified by sequence homology, Cre recombinase is among the best studied. Lox binding sites are 34 base pairs in length, but are solely sufficient to target Cre binding and recombination with the corresponding Lox sites. The canonical Lox site is the LoxP site.
  • the 8 bp spacer is asymetrical and hence has orientation (actual direction of arrow is arbitrary).
  • Two-loxP sites flanking a gene are called “floxing”. If a gene is floxed by two identical sites facing the same direction, it will be deleted with Cre recombinase. If a gene is floxed by Lox sites facing opposite directions, it will be reversed in its orientation with Cre recombinase. If two separate genes are floxed by identical sites, the genes may be exchanged with Cre recombinase. This is known as recombinase mediated cassette exchange (RMCE). Because the lox sites remain unaltered following recombination, these reactions are reversible or bidirectional.
  • RMCE recombinase mediated cassette exchange
  • This invention applies Cre recombinase and half mutant lox sites with incompatible spacers to uni-directionally exchange modified targeting genes into the fiber region of adenoviral vectors.
  • the use of a Lox m2/66 and Lox 71 on the donor fragment; and a Lox m2/71 with Lox 66 on the acceptor fragment results in a unidirectional gene exchange with maintained orientation and lack of alternative recombination events (Langer, supra).
  • the acceptor vector, pFEX has a Lox m2/66 3′ of the SacB gene and a Lox 71 on the 5′-side.
  • the donor vector, RP-Fib contains a lox m2/71 site 5′ of the Fiber gene and a Lox 66 site on the 3′-side ( FIG. 3 ).
  • the combination of the unidirectional recombination with a negative selectable marker results in extremely high numbers of desired recombinants.
  • the system is directly compatible with the existing AdEasy system.
  • the acceptor vector is similar to AdEasy-1, but it has the fiber gene replaced with a floxed negative selectable marker, the SacB gene.
  • the smaller donor vector, RP-Fib contains a modified fiber gene, which is also floxed.
  • Several variations of the smaller donor include a unique BspEI site in the HI loop for the incorporation of targeting ligands and/or a mutation in the receptor binding region of fiber. Additionally, the donor contains many convenient restriction enzyme recognition sites so genes other than fiber can be efficiently shuttled into pFex. The numerous shuttle vectors are described in detail below.
  • the fiber gene can be transferred into pFex either before ( FIG. 4 ) or after ( FIG. 5 ) the recombination with the E1 shuttle vector.
  • Two separate fiber shuttle scaffolds have been constructed for either transfer stage. RP-Fib, which is kanamycin resistant, is applied for recombination prior to the E1 shuttle recombination ( FIG. 4 ), and RPuc-Fib, which is ampicillin resistant, is applied for recombination after the E1 shuttle recombination.
  • pFex stable E. coli called bFex can be used to overcome limitations in large plasmid transformation efficiency.
  • pFex was assembled through several steps. First, a segment called ‘distal to fiber Age I’ was created by PCR amplification of the adenovirus serotype 5 genome with primers AdE-Dist 5′ and AdE-Dist 3′ (Table 1). This product was then cloned into the TOPO-TA vector pCR-2.1, using TA cloning, to produce the vector Step 1 pFex ( FIG. 6 ).
  • Step 2 pFex ( FIG. 7 ).
  • the SacB gene was isolated from the vector pAJ200 using the Bgl II and Pvu I restriction sites. Next, the two half mutant lox sites, lox m2/66 and lox 71, were added by ligation with self annealed linkers 5′ lox m2/66 and 3′ lox m2/66, and 5′ lox 71 and 3′ lox 71, respectively (Table 1). The resulting floxed SacB gene was then subcloned into Step 2 pFex to create Step 3 pFex ( FIG. 8 ).
  • pFEX ( FIG. 9 ).
  • the final product was verified by sequencing using primers pFEX for 01-11 and pFEXrev01-11 (Table 1).
  • pFEX-p* contains a mutation in the integrin binding domain of the penton gene, where RGD is mutated to RGE ( FIG. 9 ).
  • the fiber shuttle vectors were also constructed in a stepwise manner.
  • An existing adenovirus serotype 5 fiber vector, pBK-CMV-Fiber was first digested with the restriction enzymes Spe I and Xho I.
  • the linkers S-lox m2/71-X5 and S-lox m2/71-X5 were self annealed and then inserted into the vector at these sites, creating Step 1 Fiber Shuttle Lox m2/71 ( FIG. 10 ).
  • This product was then digested with restriction enzymes Acc65 I and Not I, and the linkers N-Lox 66-A-5 and N-Lox 66-A-3 (Table 2), were then ligated into this site.
  • the final product was named RP-Fib ( FIG.
  • the tripartite leader splice acceptor site was inserted downstream of the lox m2/71 site by annealing the primers splce1 (TCGAGAACTATCTTCATGTTGTTGCAGATGAAGCGCGCAAGACCGTCTGAAGATACCTTCAACCCCGTGTATC CATATGACACGGAAA) (SEQ ID NO.7) and splce 2 (CCGGTTTCCGTGTCATATGGATACACGGGGTTGAAGGTATCTTCAGACGGTCTTGCGCGCTTCATCTGCAACAACATGAAG ATAGTTC) (SEQ ID NO.8) and cloning this into XhoI/AgeI sites of all fiber shuttle vectors.
  • splce1 TCGAGAACTATCTTCATGTTGTTGCAGATGAAGCGCGCAAGACCGTCTGAAGATACCTTCAACCCCGTGTATC CATATGACACGGAAA
  • All of the described RP-Fib vectors have a mutated fiber gene that contains a unique BspEI site in the gene's HI loop for the incorporation of targeting peptide sequences ( FIG. 11 ). Additionally, some vectors have a mutated fiber gene were the coding region for T 489 AYT 492 , a known Coxsackie and Adenovirus Receptor (CAR) binding site, has been deleted (Roelvink, P. W. et al. (1999) Science, 286: 1568-1571).
  • CAR Coxsackie and Adenovirus Receptor
  • RP-Fib vectors contain genes encoding kanamycin resistance.
  • a separate set of vectors, RPuc-Fib contain the same floxed fiber genes; however, the vector base is pUC-19, which is amplicillin resistant ( FIG. 12 ). These two separate selection antibiotics allow for fiber gene exchange to occur at multiple steps ( FIGS. 4 & 5 ).
  • the pFex vector was recombined with two E1 region shuttle vectors, pAdTrack ( FIG. 13 ) and pAdTrack-CMV-Luc ( FIG. 14 ) to demonstrate working recombination in these regions.
  • This recombination step is based on the previously described AdEasy system (He, T. C. et al. (I 998) Proc Natl Acad Sci USA, 95: 2509-2514).
  • the RecA positive bacterial line BJ5183 was first stably transfected with pFex. This technique has been shown to significantly increase the number of recombinants with the AdEasy vectors (Zeng, M. et al. (2001) Biotechniques, 31: 260-262). Each pAdTrack vector was then transformed into pFex stable BJ5183 cells, followed by selection on 50 ⁇ g/ml Kanamycin.
  • the pFex vector was then recombined with the kanamycin resistant shuttle vectors Rp-Fib-1, Rp-Fib-2, Rp-Fib-3, and Rp-Fib-4 to demonstrate working Cre lox recombination.
  • This fiber exchange reaction was facilitated by the Cre expressing bacteria, 294cre.
  • pFex and molar excess of the Fiber shuttle were co-transformed into 294cre cells by electroporation. These cells were then heat shocked for 20 minutes at 42° C. to induce Cre expression, and then incubated at 37° C., while shaking, for 2 hours to continue Cre expression and Cre based recombination.
  • the pAdTrack recombination products pAdTrack-Fex and pAdTrack-Luc Fex were recombined with all four RPuc-Fib shuttle vectors.
  • the larger pAdTrack-Fex vectors were co-transformed into 294cre cells with molar excess RPuc-Fib shuttle vectors. Cre expression was induced by heat shock at 42° C. for 20 minutes, followed by 2 hour incubation at 37° C. with shaking at 225 rpm. Recombination efficiency was assessed by selection on a variety of antibiotics, with and without sucrose selection (Table 4).
  • Adenovirus containing wild type fiber was generated with pFex for the purpose of directly comparing AdEasy and pFex derived virus.
  • the E1 shuttle vector, pAdTrack was recombined into the E1 region of both AdEasy-1 and pFex.
  • the resulting pAdTrack-Fex vector was then recombined with a fiber shuttle encoding the Wild Type Fiber, Rpuc-WTFib ( FIG. 20 ).
  • the resulting pFex-based viral genome was termed “pAdTrack-WTFib”.
  • Both pAdTrack-WTFib and pAdTrack-AdEasy viral plasmids were linearized with PacI and separately transfected into 293 cells for viral production. Both plasmids generated viable virus. These were concurrently amplified, harvested, and titered. The resulting viral titers were identical between AdTrack-AdEasy and AdTrack-WTfib virus (Table 6). Further, both virus were applied to 293 cells at low multiplicity of infection (MOI) and plaques size was compared by fluorescent microscopy (GFP) to determine if there were any pFex-related deleterious effects on viral replication. Both virus had identical plaque size ( FIG. 21 ). Therefore, there appear to be no deleterious effects of the lox sites on viral production or lifecycle.
  • MOI multiplicity of infection
  • GFP fluorescent microscopy
  • the Cre-recombinase mediate fiber exchange is not limited to E. coli or plasmids.
  • Fiberless pFex viral vectors can be packaged into working virus through complementary cell lines that express wild type fiber protein (a process known as psuedotyping). These pseudotyped pFex viral vectors can then be used to infect Cre expressing mammalian cells (293cre57) that have been transfected with fiber donor vectors ( FIG. 22 ). Following recombination (2-5 days), cell lysate and supernatant are harvested and used to infect a non-Cre expressing packaging line, such as 293, 911, or 911-S11. The efficiency of recombination is such that 0.01% wild type fiber shuttle, in the background of mutant fiber shuttle, can be detected. This efficiency is great enough to generate an adenoviral peptide display library.
  • This strategy was used to generate a CAR de-targeted adenovirus, AdTrack-Fib2.
  • 293cre57 cells were simultaneously transfected with 3 ⁇ g RPuc-Fib2 ( ⁇ TAYT) and infected with pseudotyped AdTrack-Fex virus at an MOI of 1.
  • ⁇ TAYT 3 ⁇ g RPuc-Fib2
  • pseudotyped AdTrack-Fex virus 5 days post transfection-infection, cell and supernatant were harvested and freeze-thawed.
  • the resulting virus was plaque purified, amplified, and titered in 911-S11 cells.
  • the pFex system offers a unique and highly efficient means of creating fiber-modified or re-targeted adenoviral vectors.
  • This system is fully compatible with the existing AdEasy gene vector system, which is currently applied in the majority of adenoviral vector laboratories.
  • the system is very flexible, allowing Fiber gene transfer before or after E1 cassette exchange.
  • modified fiber gene can be shuttled into intact viral genomes in Cre recombinase expressing mammalian cell lines. This system is ideal for generating and screening modified fiber adenoviral vectors.
  • pFex offers a simple and efficient means to create viral vectors to reach these goals.

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Abstract

The instant invention provides methods and compositions for generating recombinant adenoviral vectors. The invention also provides kits comprising for the generation of recombinant adenoviral vectors.

Description

RELATED APPLICATIONS
This application is a continuation of PCT Application PCT/US06/10025, filed Mar. 16, 2006 which claims the benefit of U.S. Provisional Application No. 60/662,168, filed Mar. 16, 2005. The entire contents of the aforementioned applications are hereby expressly incorporated herein by reference.
GOVERNMENT SUPPORT
This work was supported, in part, by grants from the National Cancer Institute (2P50CA58236-09A1) and the Prostate Cancer Foundation (DAMD17-03-2-0033). Accordingly, the government may have certain rights to this invention.
BACKGROUND OF THE INVENTION
Recombinant adenoviruses are currently used for a variety of purposes, including gene transfer in vitro, vaccination in vivo, and gene therapy. Several features of adenovirus biology have made such viruses the vectors of choice for certain of these applications. For example, adenoviruses transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of virus and high levels of transgene expression can generally be obtained.
Decades of study of adenovirus biology have resulted in a detailed picture of the viral life cycle and the functions of the majority of viral proteins. The genome of the most commonly used human adenovirus (serotype 5) consists of a linear, 36 kb, double-stranded DNA molecule. Both strands are transcribed and nearly all transcripts are heavily spliced. Viral transcription units are conventionally referred to as early (E1, E2, E3 and E4) and late, depending on their temporal expression relative to the onset of viral DNA replication. The high density and complexity of the viral transcription units poses problems for recombinant manipulation, which is therefore usually restricted to specific regions, particularly E1, E2A, E3, and E4. In most recombinant vectors, transgenes are introduced in place of E1 or E3, the former supplied exogenously. The E1 deletion renders the viruses defective for replication and incapable of producing infectious viral particles in target cells; the E3 region encodes proteins involved in evading host immunity, and is dispensable for viral production per se.
Two approaches have traditionally been used to generate recombinant adenoviruses. The first involves direct ligation of DNA fragments of the adenoviral genome to restriction endonuclease fragments containing a transgene. The low efficiency of large fragment ligations and the scarcity of unique restriction sites have made this approach technically challenging. The second and more widely used method involves homologous recombination in mammalian cells capable of complementing defective adenoviruses (“packaging lines”). Homologous recombination results in a defective adenovirus which can replicate in the packaging line (e.g., 293 or 911 cells) which supplies the missing gene products (e.g., E1). The desired recombinants are identified by screening individual plaques generated in a lawn of packaging cells. The low efficiency of homologous recombination, the need for repeated rounds of plaque purification, and the long times required for completion of the viral production process have hampered more widespread use of adenoviral vector technology. Thus there is a need in the art for more efficient and flexible techniques for generating recombinant adenoviruses.
SUMMARY OF THE INVENTION
The instant invention provides a methods for making recombinant viral vectors and provides methods and compositions for using these vectors.
Accordingly, in one aspect, the instant invention provides methods for generating a recombinant adenoviral vector comprising a desired gene, comprising the steps of co-transforming a cell expressing RecA with a linearized shuttle plasmid comprising a selectable marker, and a transfer plasmid wherein the transfer plasmid comprises a fiber gene, thereby allowing recombination of the plasmids to generate a recombinant adenoviral vector.
In one embodiment, the transfer plasmid is constructed by co-transforming a donor plasmid and a acceptor plasmid into a cell expressing a Cre recombinase, wherein the acceptor plasmid comprises a nucleic acid segment encoding a negatively selectable marker flanked by lox sites, and a first selectable marker, and the donor plasmid comprises a nucleic acid segment encoding the fiber gene flanked by lox sites and a second selectable marker, thereby allowing for recombination of the fiber gene and the negatively selectable marker.
In a related embodiment, the lox sites are incompatible. In an further related embodiment, the lox sites are mutated to result in unidirectional recombination. In exemplary embodiments, the donor plasmid lox sites are Lox m2/66 and Lox 71 and the acceptor plasmid lox sites are Lox m2/71 and Lox 66.
In one embodiment, the negatively selectable marker is SacB.
In another embodiment, the fiber gene is modified. In one embodiment, the fiber gene is modified to incorporate a unique restriction site. In an exemplary embodiment, the unique restriction site is in the HI loop. In a further exemplary embodiment, the unique restriction site is a BspEI site.
In one embodiment, the acceptor plasmid contains a kanamycin selectable marker. In another embodiment, the donor plasmid contains a ampicillin selectable marker.
In another embodiment, the method further comprises selecting recombinant adenoviral vectors using the selectable marker. In one embodiment, the selectable marker is kanamycin.
In one embodiment, the cell is a bacterial cell, e.g., an E. coli cell. In other embodiments, the cell is a mammalian cell.
In one embodiment, the shuttle plasmid comprises a resistance gene and a nucleic acid segment encoding a desired product. In exemplary embodiments the desired product is a polypeptide or fragment thereof, a nucleic acid, a siRNA, an RNAi, an shRNA, or an aptamer. In specific exemplary embodiments, the desired product is a polypeptide, e.g., a therapeutic polypeptide.
In another embodiment, the nucleic acid segment is under control of a promoter. In a related embodiment, the promoter is a tissue specific promoter.
In another embodiment, the shuttle plasmid contains a unique restriction site located between RecA recombination sites. In an exemplary embodiment, the unique restriction site is a Pme I site. In another embodiment, this Pme I site can be used to linearize the plasmid.
In another embodiment, the shuttle plasmid further comprises RecA homologous recombination sites, e.g., Ad5 left and Ad5 right. In a related embodiment, the transfer plasmid further comprises RecA homologous recombination sites, e.g., Ad5 left and Ad5 right.
In another aspect, the instant invention provides methods for generating a recombinant adenoviral vector comprising a desired gene, comprising the steps of co-transforming a cell expressing RecA with a linearized shuttle plasmid comprising a selectable marker and a transfer plasmid wherein the transfer plasmid comprises a fiber gene, wherein the transfer plasmid is constructed by co-transforming a donor plasmid and a acceptor plasmid into a cell expressing Cre recombinase, wherein the acceptor plasmid comprises a nucleic acid segment encoding a negatively selectable marker flanked by lox sites, and a first selectable marker, and the donor plasmid comprises a nucleic acid segment encoding the fiber gene flanked by lox sites and a second selectable marker, thereby allowing for recombination of the fiber gene and the negatively selectable marker, thereby allowing recombination of the plasmids to generate a recombinant adenoviral vector.
In another aspect, the instant invention provides methods of generating a recombinant adenoviral vector comprising a desired gene, comprising the steps of co-transforming a cell expressing the Cre recombinase with a donor plasmid comprising a nucleic acid segment encoding the fiber gene flanked by lox sites and a shuttle-acceptor plasmid comprising a nucleic acid segment encoding a negatively selectable marker flanked by lox sites, and a nucleic acid segment encoding a desired product, thereby allowing recombination of the plasmids to generate a recombinant adenoviral vector.
In a related aspect, the desired product is a polypeptide, polypeptides, or fragments thereof, a nucleic acid, a siRNA, an RNAi, an shRNA, or an aptamer.
In one embodiment, the shuttle-acceptor plasmid is constructed by co-transforming a cell expressing RecA with a linearized shuttle plasmid and an acceptor plasmid comprising a negatively selectable marker.
In one embodiment, the lox sites are incompatible. In a related embodiment, the lox sites are mutated to result in unidirectional recombination. In an exemplary embodiment, the donor plasmid lox sites are Lox m2/66 and Lox 71 and the acceptor plasmid lox sites are Lox m2/71 and Lox 66.
In one embodiment, the negatively selectable marker is SacB.
In another embodiment the fiber gene is modified. In a related embodiment, the fiber gene is modified to include a unique restriction site in the HI loop. In an exemplary embodiment, the restriction site is a BspEI site.
In one embodiment, the acceptor plasmid contains a kanamycin selectable marker.
In another embodiment, the donor plasmid contains an ampicillin selectable marker.
In one embodiment, the methods further comprise selecting recombinant adenoviral vectors using the selectable marker. In an exemplary embodiment, the selectable marker is kanamycin.
In one embodiment, the cell is a bacterial cell, e.g., an E. coli cell. In another embodiment, the cell is a mammalian cell.
In one embodiment, the shuttle plasmid comprises a resistance gene and a nucleic acid segment encoding a desired product. In a related embodiment, the product is a polypeptide, polypeptides, or fragments thereof, a nucleic acid, an aptamer, a siRNA, RNAi, an shRNA, or an aptamer. In a specific embodiment, the product is a polypeptide.
In certain embodiments, the peptide is a therapeutic polypeptide.
In one embodiment, nucleic acid segment is under control of a promoter. In a related embodiment, the promoter is a tissue specific promoter.
In one embodiment, the resistance gene is not the same as the resistance gene in the acceptor plasmid.
In one embodiment, the shuttle plasmid contains a unique restriction site located between the RecA homologous recombination sites. In an exemplary embodiment, the unique restriction site is a Pme I site. In another related embodiment, the shuttle plasmid is linearized with Pme I.
In one embodiment, the acceptor plasmid further comprises RecA homologous recombination sites. In an exemplary embodiment, the RecA homologous recombination sites are Ad5 left and Ad5 right. In another embodiment, the shuttle plasmid further comprises RecA homologous recombination sites. In an exemplary embodiment, the RecA homologous recombination sites are Ad5 left and Ad5 right.
In one aspect, the invention also provides shuttle plasmids.
In another aspect the instant invention also provides acceptor plasmids.
In yet another aspect, the instant invention provides, shuttle-acceptor plasmids.
In yet another aspect, the instant invention provides donor plasmids.
In yet another aspect, the instant invention provides transfer plasmids.
In yet another aspect, the instant invention provides recombinant viral vectors comprising a resistance gene located between RecA homologous recombination sites, and a nucleic acid segment encoding a desired product.
In one embodiment, the invention provides a viral vector consisting of the nucleic acid molecule set forth as SEQ ID NO: 1.
In another embodiment, the invention provides a shuttle plasmid consisting of the nucleic acid sequence set forth as SEQ ID NO:2.
In another embodiment, the invention provides a donor plasmid consisting of the nucleic acid sequence set forth as SEQ ID NO:3.
In another embodiment, the invention provides a donor plasmid consisting of the nucleic acid sequence set forth as SEQ ID NO:4.
In another embodiment, the invention provides a donor plasmid consisting of the nucleic acid sequence set forth as SEQ ID NO: 5.
In another embodiment, the invention provides an acceptor plasmid consisting of the nucleic acid sequence set forth as SEQ ID NO:6.
In another aspect, the instant invention provides methods of creating virus comprising linearizing the viral vectors described herein and transfecting the linearized vector into a cell, thereby creating a virus.
The invention also provides methods of making psuedotyped virus using the viral vectors described herein.
In another aspect, the invention provides methods of treating an individual in need of treatment by administering to the individual a viral vector or a virus described herein.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of a lox site showing two inverted repeats separated by a spacer region (SEQ ID NO.9).
FIGS. 2A-B depict various lox sequences. FIG. 2A depicts lox sequences with half-site mutations in italics. FIG. 2B depicts spacer sequences with mutations in italics (SEQ ID NOS.9 and 11-13).
FIG. 3 is a schematic depicting two non-compatible spacer sequences (black arrows) that force gene exchange rather than excision. The reaction of two half-mutant lox sites (shaded with mutations in lower case) results in a dually mutated lox site (PR-SacB) and a unidirectional reaction (SEQ ID NOS.12-15).
FIG. 4 is a schematic of pFex fiber exchange followed by RecA recombination resulting in pShuffle-Fib, an adenoviral vector. This vector can be digested with Pac I and transfected into a desired cell line to create virus.
FIG. 5 is a schematic of Rec A recombination followed by pFex fiber exchange. The pshuttle-Fib is the completed adenoviral vector. This vector can be digested with Pac I and transfected into a desired cell line to create virus.
FIG. 6 is a schematic depicting step 1 of pFex assembly.
FIG. 7 is a schematic depicting step 2 of pFex assembly.
FIG. 8 is a schematic depicting step 3 of pFex assembly.
FIG. 9 is a schematic showing vectors pFex and pFex-p*.
FIG. 10 is a schematic depicting Fiber Shuttle Lox m2/71.
FIG. 11 is a schematic of RP-Fib-1.
FIG. 12 is a schematic of RPuc-Fib-1.
FIG. 13 is a schematic of pAdTrack shuttle vector.
FIG. 14 is a schematic of pAdTrack-CMV-Luc shuttle vector.
FIG. 15 depicts the results of restriction digests demonstrating pFex recombination with pAdTrack vectors.
FIG. 16 depicts the results of restriction digests demonstrating ColE1/Ad Right hand recombination.
FIG. 17 depicts the results of restriction digests indicating the expected products by co-transformation of fiber shuttle and pFex into 294cre cells or by transformation of fiber shuttle into pFex stable 294cre cells (b294-fex).
FIG. 18 depicts the results of restriction digests indicating that transformants contained the desired products.
FIG. 19 depicts the results of restriction digests indicating that all products have the expected molecular weight. Track-Fib refers to pAdTrack recombinants and Luc-Fib refers to the pAdTrack-Luc-Fib recombinants.
FIG. 20 is a schematic of Rpuc-WTFib, a fiber shuttle that contains wild-type fiber cDNA.
FIG. 21 depicts a comparison of the plaque size of AdTrack-AdEasy and AdTrack-WTFib by fluorescent microscopy.
FIG. 22 depicts CRE mediated fiber exchange into viral genome in mammalian 293-cre cells through the use of a pseudotyped pAdTrack-pFex virus.
FIG. 23 depicts detargeted AdTrack-Fib2 virus which was generated by pseudotyped AdTrack-Fex recombination with Rpuc-Fib2 in Cre recombinase expressing mammalian cells.
FIG. 24 is Table 1 entitled Primers for Constructing and Sequencing pFex (SEQ ID NOS.19-48).
FIG. 25 is Table 2 entitled Primers for Constructing and Sequencing Fiber Shuttles (SEQ ID NOS.49-52).
FIG. 26 is Table 3 entitled Fiber Shuttle Vectors.
FIG. 27 is Table 4 entitled Total Transformants from 294 co-transfections.
FIG. 28 is Table 5 entitled Percent Recombinants of Large pAdTrack-Fex Vector.
FIG. 29 is Table 6 entitled pFex and AdEasy based virus.
FIG. 30 depicts the nucleic acid sequence of pShuttle-Fib (SEQ ID NO: 1).
FIG. 31 depicts the nucleic acid sequence of pShuttle (SEQ ID NO:2).
FIG. 32 depicts the nucleic acid sequence of RP-Fib (SEQ ID NO:3).
FIG. 33 depicts the nucleic acid sequence of RPuc-Fib (SEQ ID NO:4).
FIG. 34 depicts the nucleic acid sequence of RP-Blast-Fib (SEQ ID NO: 5).
FIG. 35 depicts the nucleic acid sequence of pFEX (SEQ ID NO:6).
DETAILED DESCRIPTION OF THE INVENTION
The instant invention is based, at least in part, on the discovery of methods for generating adenoviral vectors which are more efficient and more flexible than current systems for producing recombinant viral vectors. These methods and vectors are compatible with current technology as described in U.S. Pat. No. 5,922,576, the contents of which is expressly incorporated herein by reference.
Though several systems for generating recombinant viruses through Cre-mediated or homologous recombination in yeast or bacteria have been described in the literature, the instant invention provides a two-stage recombination system for making recombinant adenoviral vectors that has several advantages in terms of ease, sensitivity and flexibility. The instant invention provides methods of producing recombinant adenoviral vectors that offer the practitioner the flexibility to adapt the system to their particular needs. They skilled artisan may introduce the desired product into an already completed transfer vector and select for a recombinant adenoviral vector, or introduce the desired product into the acceptor vector as the initial step in the method, thereby allowing them the flexibility that other systems do not provide. Moreover, the ability to recover very small numbers of recombinant viral particles amongst many transformants is particularly advantageous.
Publications describing various aspects of adenovirus biology and/or techniques relating to adenovirus include the following. Graham and Van de Eb (1973) Virology 52:456-467; Takiff et al. (1981) Lancet ii:832-834; Berkner and Sharp (1983) Nucleic Acid Research 6003-6020; Graham (1984) EMBO J. 3:2917-2922; Bett et al. (1993) J. Virology 67:5911-5921; and Bett et al. (1994) Proc. Natl. Acad. Sci. USA 91:8802-8806 describe adenoviruses that have been genetically modified to produce replication-defective gene transfer vehicles. In these vehicles, the early adenovirus gene products E1A and E1B are deleted and provided in trans by the packaging cell line 293 developed by Frank Graham (Graham et al. (1987) J. Gen. Birol. 36:59-72 and Graham (1977) J. Genetic Virology 68:937-940). The gene to be transduced is commonly inserted into adenovirus in the deleted E1A and E1B region of the virus genome Bett et al. (1994), supra. Adenovirus vectors as vehicles for efficient transduction of genes have been described by Stratford-Perricaudet (1990) Human Gene Therapy 1:2-256; Rosenfeld (1991) Science 252:431-434; Wang et al. (1991) Adv. Exp. Med. Biol. 309:61-66; Jaffe et al. (1992) Nat. Gent. 1:372-378; Quantin et al. (1992) Proc Natl. Acad. Sci. USA 89:2581-2584; Rosenfeld et al. (1992) Cell 68:143-155; Stratford-Perricaudet et al. (1992) J. Clin. Invest. 90:626-630; Le Gal La Salle et al. (1993) Science 259:988-990; Mastrangeli et al. (1993) J. Clin. Invest. 91:225-234; Ragot et al. (1993) Nature 361:647-650; Hayaski et al. (1994) J. Biol. Chem. 269:23872-23875.
The present invention utilizes recombination, e.g., recombination in bacteria, to combine plasmid DNA molecules containing a desired product to form an adenoviral vector. In specific embodiments, the instant invention provides methods for generating recombinant adenoviral vectors that utilizes RecA and Cre mediated homologous recombination. Recombination is a process in which two DNA molecules become joined and nucleic acid is exchanged. Homologous recombination occurs between two sequences having regions of homology. Bacterial recombination is particularly robust. In order to facilitate recombination between the DNA molecules, i.e., plasmids, identical sequences must be present in both. Using standard methods in the art, segments of the adenoviral genome can be put in the plasmids to create regions of homology.
An “adenovirus vector” or “adenoviral vector” (used interchangeably) is a term well understood in the art and generally comprises a polynucleotide comprising all or a portion of an adenovirus genome. As used herein, “adenovirus” refers to the virus itself or derivatives thereof. The term covers all serotypes and subtypes and both naturally occurring and recombinant forms, except where otherwise indicated. An adenoviral vector of the present invention can be in any of several forms, including, but not limited to, naked DNA; an adenoviral vector encapsulated in an adenovirus coat; packaged in another viral or viral-like form (such as herpes simplex virus and AAV); encapsulated in a liposome; complexed with polylysine or other biocompatible polymer; complexed with synthetic polycationic molecules; conjugated with transferrin; complexed with compounds such as PEG to immunologically “mask” the molecule and/or increase half-life, or conjugated to a non-viral protein. An adenoviral vector of this invention may be in the form of any of the delivery vehicles described herein. Such vectors are one embodiment of the invention. Preferably, the polynucleotide is DNA. As used herein, “DNA” includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides. For purposes of this invention, adenovirus vectors are replication-competent in a target cell.
The term “plasmid” denotes an extrachromosomal circular DNA capable of autonomous replication in a given cell. The range of suitable plasmids is very large. Preferably, the plasmid is designed for amplification in bacteria and for expression in an eukaryotic target cell. Such plasmids can be purchased from a variety of manufacturers. Exemplary plasmids include but are not limited to those derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), pREP4, pCEP4 (Invitrogene), pCI (Promega) and p Poly (Lathe et al., Gene 57 (1987), 193-201). Plasmids can also be engineered by standard molecular biology techniques (Sambrook et al., Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), N.Y.). It may also comprise a selection gene in order to select or to identify the transfected cells (e.g., by complementation of a cell auxotrophy or by antibiotic resistance), stabilizing elements (e.g., cer sequence; Summers and Sherrat, 1984, Cell 36, 1097-1103) or integrative elements (e.g., LTR viral sequences and transposons).
As used herein the term “shuttle plasmid” is intended to mean a plasmid comprising a unique restriction site between RecA homologous recombination sites and used to insert a desired nucleic acid molecule, i.e., a nucleic acid molecule encoding a desired product, into a recombinant adenoviral vector. The RecA homologous recombination sites can be, for example, Ad5 right and Ad5 left. In further embodiments, the shuttle plasmid may have a tissue specific promoter which controls the expression of the desired nucleic acid molecule. The shuttle plasmid also contains a majority of the viral genes necessary to form viral particles. However, the shuttle plasmid does not contain all necessary genes to form viral particles. An exemplary shuttle plasmid is referred to as pShuttle herein.
As used herein, RecA mediated homologous recombination is used to exemplify enzyme mediated homologous recombination. Other enzymes capable of mediating homolgous recombination are known in the art and can be used to design the vectors of the invention, and can further be used in the methods of the invention. For example, homologous recombination enzymes are known in eukaryotes, e.g., Rad51, Rad57, Rad55 and DMC1, in Archaea, e.g., RadA and RadB, and in phage, e.g., vsX in phage T4. These enzymes and homologs and orthologs of these enzymes are envisioned for use in the methods of the present invention.
As used herein the term “transfer plasmid” is intended to mean the plasmid that results from the Cre mediated recombination of the donor plasmid and the acceptor plasmid. The transfer plasmid has the fiber gene, or other gene in the fiber location, inserted in place of the negatively selectable marker. Moreover, the transfer plasmid has RecA homologous recombination sites to allow for insertion of a desired nucleic acid molecule by RecA mediated homologous recombination with the shuttle plasmid. The transfer plasmid also has a selectable marker, i.e., ampicillin located between the RecA homologous recombination sites. The RecA homologous recombination sites can be, for example, Ad5 right and Ad5 left. An exemplary transfer plasmid is referred to as pFex-Fib herein.
As used herein the term “nucleic acid molecule encoding fiber” is intended to mean a nucleic acid segment encoding viral capsid protein that is responsible for mediating high-affinity attachment of adenovirus to a target cell. The amino acid sequence of fiber is available as GenBank Accession number P03275, and is further described by Herisse, J., et al. (1981) Nucleic Acids Res. 9:4023-4042. In specific embodiments, the fiber gene used in the methods and compositions of the invention can be a functional fragment of the fiber protein, i.e., a fragment that retains the ability to allow the attachment of a virus to a cell.
As used herein the term “donor plasmid” is intended to mean a plasmid containing a donor gene flanked on either side by lox sites. In exemplary embodiments of the invention the donor gene is a fiber gene, or fragment thereof. However, one skilled in the art would understand that other genes can be used in place of fiber. For example, another gene that encodes a cell surface recognition protein can be used in place of fiber. Also, a nucleic acid molecule encoding a toxin can be used in place of fiber. In order to select for the transfer plasmid, the donor plasmid has a different selectable marker than the acceptor plasmid. In exemplary embodiments, the donor plasmid has ampicillin, kanamycin, or blastocidin resistance. Exemplary donor plasmids are referred to as RP-Fib, RPuc-Fib, and Rblast-Fib herein.
As used herein the term “acceptor plasmid” is intended to mean a plasmid containing a negatively selectable marker flanked by lox sites and a selectable marker, e.g., ampicillin, located between RecA homologous recombination sites. The negatively selectable marker can be, for example, SacB. An exemplary acceptor plasmid is referred to as pFex herein.
As used herein the term “shuttle-acceptor plasmid” is intended to mean the recombination product of RecA mediated recombination of a shuttle plasmid and an acceptor plasmid. The shuttle-acceptor plasmids of the invention comprise a negatively selectable marker located between two lox sites, a resistance marker, and a nucleic acid molecule encoding a desired product. An exemplary shuttle-acceptor plasmid is referred to as pShuttle-Fex herein.
In one embodiment, the “desired product” in use in the present invention, encodes a gene product of therapeutic interest. A “desired product” can have a therapeutic or protective activity when administered appropriately to a patient, especially a patient suffering from a disease or illness condition or who should be protected against this disease or condition. Such a therapeutic or protective activity can be correlated to a beneficial effect on the course of a symptom of said disease or said condition. It is within the reach of the man skilled in the art to select a gene encoding an appropriate gene product of therapeutic interest, depending on the disease or condition to be treated. In a general manner, his choice may be based on the results previously obtained, so that he can reasonably expect, without undue experimentation, i.e., other than practicing the invention as claimed, to obtain such therapeutic properties.
In the context of the invention, the desired product can be homologous or heterologous to the host cell into which it is introduced. Advantageously, it encodes a polypeptide, a ribozyme or anti-sense RNA, RNAi, an aptamer or the like. The term “polypeptide” is to be understood as any translational product of a polynucleotide whatever its size is, and includes polypeptides having as few as 7 residues (peptides), but more typically proteins. In addition, it may be from any origin (prokaryotes, lower or higher eukaryotes, plant, virus etc). It may be a native polypeptide, a variant, a chimeric polypeptide having no counterpart in nature or fragments thereof. Advantageously, the gene of interest in use in the present invention encodes at least one polypeptide that can compensate for one or more defective or deficient cellular proteins in an animal or a human organism, or that acts through toxic effects to limit or remove harmful cells from the body. A suitable polypeptide may also be immunity conferring and acts as an antigen to provoke a humoral or a cellular response, or both.
The regulatory elements controlling the expression of the desired gene may further comprise additional elements, such as exon/intron sequences, targeting sequences, transport sequences, secretion signal sequences, nuclear localization signal sequences, IRES, polyA transcription termination sequences, tripartite leader sequences, sequences involved in replication or integration. These elements have been reported in the literature and can be readily obtained by those skilled in the art.
As used herein the term “lox sites” is intended to mean a nucleic acid sequence that the Cre recombinase recognizes. The canonical lox site is the loxP site. Lox sites are 34 nucleotides in length and have a 13 base pair inverted repeat separated by an 8 base pair spacer (see FIG. 1). Wild-type lox sites are unaltered following recombination thereby allowing for a reversible reaction. The instant invention uses “incompatible” lox sites which have a mutation such that intrageneic recombination, i.e. recombination within a plasmid which can result in deletion or inversion of flanked nucleic acid, can not occur. Exemplary mutations include those to the spacer that result in non-functional lox sites following recombination (see FIGS. 2A-B). The instant invention also applies “half-mutant” lox sites, which when correctly recombined, produce one fully mutant lox site and one wild type lox site, resulting in a non-functional lox site, thus preventing the reverse reaction. Specific exemplary incompatible lox sites for uni-directional insertion include the Lox m2/66 and Lox 71 on the donor fragment and Lox m2/71 with Lox66 on the acceptor fragment (see, for example, Langer, S. J. et al. (2002) Nucleic Acid Research 20:3067-77)
The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support. Preferably, the polynucleotide is DNA. As used herein, “DNA” includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides.
A key step in the generation of adenoviral plasmids according to the present invention is the co-transformation of bacteria with precursor DNA vectors. Transformation is the introduction of DNA into a bacterial cell. Transformation can be carried out by a number of techniques known in the art. Such methods include but are not limited to electroporation (exposure of a cell suspension to an electrical field), the use of calcium phosphate solutions, and the use of lipids to package the DNA and fuse with the cell membrane. Co-transformation refers to the introduction of two different species of DNA molecule into the same cell.
The plasmid desirably comprises one or more desired product. In addition, segments of DNA consisting of adenoviral sequences flank the desired product to promote homologous recombination with other nucleic acid molecules to ultimately produce an adenoviral vector.
The adenoviral vector typically contains most of the adenoviral genome. The adenoviral vector may also contain a bacterial origin of replication. Portions of the wild-type adenoviral genome may be deleted to permit insertion of desired products and the packaging of recombinant adenoviral vectors containing the desired genes.
The invention provides alternative methods for producing recombinant adenoviral vectors. The methods rely on two homologous recombination steps, one mediated by Cre and the other mediated by RecA. In alternate embodiments, the instant invention provides methods in which the Cre mediated recombination must precede the RecA recombination, methods in which the RecA mediated recombination must precede the Cre mediated recombination, and finally methods in which the order of recombination events in immaterial. The order of recombination events is dictated by the resistance genes on the precursor plasmids. For example, if the donor and shuttle plasmids have the same resistance gene, the Cre mediated recombination must be preformed first (see, for example, the schematic set forth in FIG. 4). In an alternate embodiment, if the donor and acceptor plasmids have the same resistance gene, the RecA mediated recombination must occur first (see, for example, the schematic set forth in FIG. 5). Lastly, if the donor has a different resistance gene than both the acceptor and shuttle plasmids, the order of recombination steps is at the discretion of the skilled artisan (for example, if the donor plasmid had blasticidin resistance as described in the examples).
In one embodiment, a Cre expressing cell is transformed with a donor and acceptor plasmid such that Cre mediated recombination results in the formation of a transfer plasmid. The donor plasmid contains a fiber gene, or other gene product to target the recombinant virus to a specific cell, flanked by lox sites. The acceptor plasmid has a negatively selectable marker, such as SacB, flanked by lox sites. In preferred embodiments of the invention, the lox sites are engineered, i.e., mutated, to result in irreversible, uni-directional recombination and to prevent intragenic recombination.
Cells containing the recombinant transfer plasmid are selected by growth in media containing a substrate for the negatively selectable marker and an antibiotic for which the resulting transfer plasmid carries a resistance gene. In exemplary embodiments, the negatively selectable gene is SacB and the antibiotic resistance is to ampicillin, and cells containing the recombinant transfer plasmid are selected by growth in media containing sucrose and ampicillin. Once cells containing transfer plasmids are isolated, the transfer plasmids can be isolated and transformed into a RecA expressing cell with linear shuttle plasmids. Linear shuttle plasmids are formed by digesting shuttle plasmids with one or more restriction enzymes. In one embodiment, the shuttle plasmid is linearized using a restriction enzyme that has a single restriction site in the plasmid. Alternatively, shuttle plasmids may not be linearized prior to introducing them into a cell for recombination. Recombinant adenoviral vectors formed as a result of RecA mediated recombination are selected by growing cells in the presence of an antibiotic which the recombinant adenoviral vectors carry a resistance gene against. This resistance gene was originally contained on the shuttle plasmid and is integrated into the recombinant viral vector during RecA mediated recombination. A schematic of this embodiment is set forth in FIG. 4.
In an alternate embodiment, the recombinant viral vectors are produced by transforming a cell expressing RecA with a linear shuttle plasmid and an acceptor plasmid. Cells containing a shuttle-acceptor plasmid are selected in media containing an antibiotic to which the resulting shuttle-acceptor plasmid confers resistance. Recombinant shuttle-acceptor plasmids are isolated and transformed into a cell expressing Cre along with a donor plasmid. Recombinant adenoviral vectors are selected using by growing cells in media containing a substrate for the negatively selectable marker and an antibiotic which recombinant adenoviral vectors carry a resistance gene against. This resistance gene was originally contained on the donor plasmid and is integrated into the recombinant viral vector during Cre mediated recombination. A schematic of this embodiment is set forth in FIG. 5.
In other embodiments, the Cre-recombinase mediate exchange is not limited to bacteria or plasmids. For example, fiberless acceptor plasmids can be packaged into working virus through complementary cell lines that express fiber protein (a process known as psuedotyping). These pseudotyped acceptor plasmids can then be used to infect Cre expressing cells, e.g., mammalian cells such as 293cre57, that have been transfected with fiber exchange vectors, i.e. donor vectors. Cell lysate and supernatant are then harvested and used to infect a non-Cre expressing packaging line, immediately generating a recombinant adenovirus.
Adenoviral particles can be prepared according to any conventional technique in the field of the art, such as homologous recombination in a permissive cell line (e.g., as described in Graham and Prevect, 1991, Methods in Molecular Biology, Vol 7, Gene Transfer and Expression Protocols; Ed E. J. Murray, The Human Press Inc, Clinton, N.J.) or in Escherichia coli (as described in WO96/17070). Propagation is advantageously performed in a complementing cell line or in the presence of a helper virus providing complementation in trans. “Complementing” or “complementation” denotes that the capability to encode and/or express functions that are defective in the vector but necessary for generating viable viral particles. The cell lines 293 (Graham et al., 1977, J. Gen. Virol. 36, 59-72) and PERC6 (Fallaux et al., 1998, Human Gene Therapy 9, 1909-1917) are commonly used to complement the E1 function. Other cell lines have been engineered to complement doubly defective vectors (Yeh et al., 1996, J. Virol. 70, 559-565; Krougliak and Graham, 1995, Human Gene Ther. 6, 1575-1586; Wang et al., 1995, Gene Ther. 2, 775-783; Lusky et al., 1998, J. Virol. 72, 2022-2033; EP919627 and WO97/04119). The adenoviral particles can be recovered from the culture supernatant but also from the cells after lysis and optionally further purified according to standard techniques (e.g., chromatography, ultracentrifugation, as described in WO96/27677, WO98/00524 and WO98/26048). Furthermore, the virions may be amplified by successive passage in a permissive cell in order to generate a high titer viral stock that may be used in the preparation of clinical lots.
The recombinant adenovirus vector generated as described above may be used to transfect mammalian cells. Techniques for transfection are well known. Available techniques include but are not limited to electroporation, the use of calcium chloride, and packaging of the vector together with lipid for fusion with the cells of interest. Cells may be transfected with the vector either in vitro or in vivo. The design of the recombinant adenoviral vector may place specific constraints on cells to be transfected. If production of viral particles is desired, a special packaging cell must be used that produces the adenoviral gene products which the adenoviral vector lacks. Which packaging cells are employed to replicate the virus will depend on the composition of the adenoviral vector used. The adenoviral vector may have specific portions of the adenoviral genome deleted, in order to make room for the desired gene in the recombinant vector. Suitable deletions which may be used include those of all or part of adenoviral transcription units E1, E3, and E4. The packaging cells preferably stably express the adenoviral proteins coded by the deleted transcription units. Techniques are known in the art for stably transfecting a cell line with whichever adenoviral sequences are required, i.e., by incorporation of the genes into the cell's genome. If virus particle production is not required, then packaging cell lines need not be used. For example, if cells are to express the desired product, production of viral particles need not be achieved. Thus for in vivo gene therapy, the recipient cells need not be able to complement the defective viruses.
Genes encoding a detectable marker may be present in adenoviral vector to allow for detection of the recombinant vector once produced. Preferably, a marker is used which is easy to monitor. More preferably a marker is used which can be detected even when present at very low levels. Use of a detectable marker permits monitoring of the transfection process. In an exemplary embodiment the detectable marker is β-galactosidase or green fluorescent protein (GFP). Detection of GFP can be achieved, for example, by fluorescence microscopy of cultured cells.
Genes encoding a selectable product can also be used as linked markers to the desired product. A selectable product is necessary for growth under a particular set of conditions. Thus it can be used to selectively grow only those cells that have been transformed or transfected. A preferred selectable product is an antibiotic resistance enzyme, such as those for ampicillin, kanamycin, or blastocidin.
The adenoviral vector of the invention can also be used to produce a pseudotyped viral particle, i.e., a viral particle that contains one or more structural genes that are not derived from the adenoviral genome. The viral vectors described herein can be made by recombination in intact viral genomes thereby producing pseudotyped virus.
Cell type-specific targeting may be achieved with vectors derived from viruses having a broad host range by the modification of viral surface proteins. For example, the specificity of infection of adenoviruses is determined by the attachment to cellular receptors present at the surface of permissive cells. In this regard, the fiber gene is exemplified throughout the instant application. However, those of skill in the art will recognize that many other genes can be used in place of fiber to achieve cell-type specific targeting. For example, penton plays a critical role in cellular attachment (Defer et al. J. Virol. 64 (1990) 3661-3673). Thus, cell targeting of adenoviruses can be carried out by genetic modification of a viral gene, e.g., fiber and/or penton, to generate modified proteins capable of specific interaction with unique cell surface polypeptides. Examples of such modifications are described in literature (for example in Wickam et al., 1997, J. Virol. 71, 8221-8229; Arnberg et al., 1997, Virol. 227, 239-244; Michael et al., 1995, Gene Therapy 2, 660-668; WO94/10323). Moreover, a exemplary penton mutant is described herein and called pFex-p* (mutation D342E).
The present invention also provides a host cell comprising an adenoviral vector of the invention, a polynucleotide or an expression vector as defined in connection with the use of the invention or infected by a viral particle of the invention. The vector may be inserted into the cellular genome or not (episome). A host cell may be unique type of cells or a group of different types of cells and encompass cultured cell lines, primary cells and proliferative cells, with a special preference for cells of human origin.
The present invention also provides compositions, e.g., pharmaceutical compositions, comprising as an agent an adenoviral vector according to the invention, a polynucleotide or an expression vector as described in connection with the use of the invention, a host cell or a viral particle according to the invention or prepared according to the method of the invention.
The composition according to the invention may be manufactured in a conventional manner for a variety of modes of administration including systemic, topical and local administration. Referring to systemic administration, injection is preferred, e.g., intravenous, intraperitoneal, intragastric, subcutaneous, intracardiac, intraarterial, intracoronary, intravascular, intraarterial, intramuscular, intrathecal, intratumoral, intranasal, intrapulmonary or intratracheal routes. Local administration include aerosolization instillation and oral routes of administration. The administration may take place in a single dose or a dose repeated one or several times after a certain time interval. The appropriate administration route and dosage vary in accordance with various parameters, for example, with the individual, the condition or disease to be treated, the stage to which it has progressed, the need for prevention or therapy and the gene of interest to be transferred. As an indication, a composition based on viral particles may be formulated in the form of doses of between 104 and 1014 iu (infectious unit), advantageously between 105 and 1013 iu and preferably between 106 and 1012 iu. The titer may be determined by conventional techniques. The doses of DNA vector are preferably comprised between 0.01 and 10 mg/kg, and more especially between 0.5 and 2 mg/kg. The composition of the invention can be in various forms, e.g., solid (powder, lyophilized form) or liquid (e.g., aqueous).
In a preferred embodiment, the composition comprises a pharmaceutically acceptable carrier, allowing its use in a method for the therapeutic treatment of humans or animals. In this particular case, the carrier is preferably a pharmaceutically suitable injectable carrier or diluent which is non-toxic to a human or animal organism at the dosage and concentration employed (for examples, see Remington's Pharmaceutical Sciences, 16.sup.th ed. 1980, Mack Publishing Co). It is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength, such as provided by a sucrose solution. Furthermore, it may contain any relevant solvents, aqueous or partly aqueous liquid carriers comprising sterile, pyrogen-free water, dispersion media, coatings, and equivalents, or diluents (e.g., Tris-HCl, acetate, phosphate), emulsifiers, solubilizers, excipients or adjuvants. The pH of the composition is suitably adjusted and buffered in order to be appropriate for use in humans or animals. Representative examples of carriers or diluents for an injectable composition include water, isotonic saline solutions which are preferably buffered at a physiological pH (such as phosphate buffered saline, Tris buffered saline, mannitol, dextrose, glycerol containing or not polypeptides or proteins such as human serum albumin). For example, such a composition may comprise 10 mg/ml mannitol, 1 mg/ml HSA, 20 mM Tris pH 7.2 and 150 mM NaCl.
In addition, the composition according to the present invention may include one or more stabilizing substance(s), such as lipids (e.g., cationic lipids, liposomes, lipids as described in WO98/44143; Felgner et al., 1987, Proc. West. Pharmacol. Soc. 32, 115-121; Hodgson and Solaiman, 1996, Nature Biotechnology 14, 339-342; Remy et al., 1994, Bioconjugate Chemistry 5, 647-654), nuclease inhibitors, hydrogel, hyaluronidase (WO98/53853), collagenase, polymers, chelating agents (EP890362), in order to preserve its degradation within the animal/human body and/or improve delivery into the host cell. Such substances may be used alone or in combination (e.g., cationic and neutral lipids). It may also comprise substances susceptible to facilitate gene transfer for special applications, such as a gel complex of polylysine and lactose facilitating delivery by intraarterial route (Midoux et al., 1993, Nucleic Acid Res. 21, 871-878) or poloxamer 407 (Pastore, 1994, Circulation 90, 1-517). It has also be shown that adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells. The mixture of adenoviruses to solutions containing a lipid-complexed plasmid vector or the binding of DNA to polylysine covalently attached to adenoviruses using protein cross-linking agents may substantially improve the uptake and expression of the vector (Curiel et al., 1992, Am. J. Respir. Cell. Mol. Biol. 6, 247-252).
The present invention also provides the use of an adenoviral vector according to the invention, a polynucleotide or an expression vector, as described in connection with the use according to the invention, a viral particle or a host cell according to the invention for the preparation of a medicament intended for gene transfer, preferably into a human or animal body. Within the scope of the present invention, “gene transfer” has to be understood as a method for introducing any gene of interest into a cell. Thus, it also includes immunotherapy that relates to the introduction of a potentially antigenic epitope into a cell to induce an immune response which can be cellular or humoral or both.
For this purpose, the adenoviral vector, the polynucleotide and expression vector or the viral particle of the present invention may be delivered in vivo to the human or animal organism by specific delivery means adapted to the pathology to be treated. For example, a balloon catheter or a stent coated with the adenoviral vector, the expression vector carrying the polynucleotide or the viral particle may be employed to efficiently reach the cardiovascular system (as described in Riessen et al., 1993, Hum Gene Ther. 4, 749-758; Feldman and Steg, 1996, Medecine/Science 12, 47-55). It is also possible to deliver said therapeutic agents by direct administration, e.g., intravenously, in an accessible tumor, in the lungs by aerosolization and the like. Alternatively, one may employ eukaryotic host cells that have been engineered ex vivo to contain the adenoviral vector, the expression vector carrying the polynucleotide or the viral particle according to the invention. Methods for introducing such elements into an eukaryotic cell are well known to those skilled in the art and include microinjection of minute amounts of DNA into the nucleus of a cell (Capechi et al., 1980, Cell 22, 479-488), transfection with CaPO.sub.4 (Chen and Okayama, 1987, Mol. Cell. Biol. 7, 2745-2752), electroporation (Chu et al., 1987, Nucleic Acid Res. 15, 1311-1326), lipofection/liposome fusion (Felgner et al., 1987, Proc. Natl. Acad. Sci. USA 84, 7413-7417) and particle bombardement (Yang et al., 1990, Proc. Natl. Acad. Sci. USA 87, 9568-9572). The graft of engineered cells is also possible in the context of the present invention (Lynch et al, 1992, Proc. Natl. Acad. Sci. USA 89, 1138-1142).
The present invention also relates to a method for the treatment of a human or animal organism, comprising administering to said organism a therapeutically effective amount of an adenoviral vector of the invention, the polynucleotide or expression vector as described in connection with the use according to the invention, a viral particle or an eukaryotic cell according to the invention.
A “therapeutically effective amount” is a dose sufficient for the alleviation of one or more symptoms normally associated with the disease or condition desired to be treated. When prophylactic use is concerned, this term means a dose sufficient to prevent or to delay the establishment of a disease or condition.
The method of the present invention can be used for preventive purposes and for therapeutic applications relative to the diseases or conditions listed above. The present method is particularly useful to prevent or reduce the establishment of an inflammatory response following administration of a conventional gene-therapy vector. It is to be understood that the present method can be carried out by any of a variety of approaches. Advantageously, the vector, viral particle, cell or the pharmaceutical composition of the invention can be administered directly in vivo by any conventional and physiologically acceptable administration route, for example by intravenous injection, by direct injection into an accessible tumor or by means of an appropriate catheter into the vascular system, etc. Alternatively, the ex vivo approach may also be adopted which consists of introducing the adenoviral vector, the polynucleotide or the viral particle according to the invention into cells, growing the transfected/infected cells in vitro and then reintroducing them into the patient to be treated.
A kit according to the invention comprises one or more of the described plasmids, e.g., a shuttle plasmid, a transfer plasmid, a donor plasmid, and/or an acceptor plasmid, useful in the generation of recombinant adenoviral vectors. A user of the kit may insert one or more desired genes into the shuttle plasmid using, for example, a restriction endonuclease and a DNA ligase. The kit may also comprise a packaging cell line for producing virus particles from the defective adenoviral vector and/or the recombinant adenoviral vectors produced containing the desired product. The kit may also comprise bacterial cells which can be used for co-transformation. Preferably the bacterial cells are homologous-recombination proficient and highly competent to receive transforming DNA. Typically, each kit component is separately packaged to avoid premature mixing. Further, all individually packaged components are provided in a box or other container which holds the other components. Instructions for making a recombinant adenovirus vector according to the methods disclosed herein may also be included in the kit. Reference to instructions may also be provided in the kit, for example to a text or webpage.
Kits may also contain the recombinant adenoviral vectors, or viral particles, produced by the methods of the invention and instructions for the administration of the vectors or viral particles to a subject for therapeutic or preventative purposes.
EXAMPLES
It should be appreciated that the invention should not be construed to be limited to the examples that are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.
Example 1
The Cre recombinase from bacteriophage P1 is an enzyme which mediates the excision and integration of DNA based on specific sequence binding sites (lox) through stepwise cleavage and ligation involving Holiday Junction intermediates (Ghosh, K. and Van Duyne, G. D. (2002) Methods, 28: 374-383). Though nearly 100 related tyrosine recombinases have been identified by sequence homology, Cre recombinase is among the best studied. Lox binding sites are 34 base pairs in length, but are solely sufficient to target Cre binding and recombination with the corresponding Lox sites. The canonical Lox site is the LoxP site. It has a 13 bp inverted repeat and an 8 bp spacer (FIG. 1). The 8 bp spacer is asymetrical and hence has orientation (actual direction of arrow is arbitrary). Two-loxP sites flanking a gene are called “floxing”. If a gene is floxed by two identical sites facing the same direction, it will be deleted with Cre recombinase. If a gene is floxed by Lox sites facing opposite directions, it will be reversed in its orientation with Cre recombinase. If two separate genes are floxed by identical sites, the genes may be exchanged with Cre recombinase. This is known as recombinase mediated cassette exchange (RMCE). Because the lox sites remain unaltered following recombination, these reactions are reversible or bidirectional.
In order to maximize gene replacement, without favoring spontaneous excision, two Lox sites have to be used which are incompatible. This can be accomplished by mutating the spacer (FIG. 2B). In addition, half site mutations in the inverted repeat section can lead to a unidirectional recombination event by resulting in a non-functional lox site following recombination (FIG. 2A). By combining these two methods, a highly efficient unidirectional gene replacement can be achieved (Langer, S. J. et al. (2002) Nucleic Acids Res, 30: 3067-3077.).
This invention applies Cre recombinase and half mutant lox sites with incompatible spacers to uni-directionally exchange modified targeting genes into the fiber region of adenoviral vectors. As delineated by Langer et al, the use of a Lox m2/66 and Lox 71 on the donor fragment; and a Lox m2/71 with Lox 66 on the acceptor fragment results in a unidirectional gene exchange with maintained orientation and lack of alternative recombination events (Langer, supra). Here the acceptor vector, pFEX, has a Lox m2/66 3′ of the SacB gene and a Lox 71 on the 5′-side. When induced by media containing 5% sucrose, SacB is lethal in a wide range of Gram negative bacteria, and thus permits selection for loss of the vector (Quandt, J. and Hynes, M. F. (1993) Gene, 127: 15-21). The donor vector, RP-Fib, contains a lox m2/71 site 5′ of the Fiber gene and a Lox 66 site on the 3′-side (FIG. 3). The combination of the unidirectional recombination with a negative selectable marker results in extremely high numbers of desired recombinants. The system is directly compatible with the existing AdEasy system. The acceptor vector, named pFex, is similar to AdEasy-1, but it has the fiber gene replaced with a floxed negative selectable marker, the SacB gene. The smaller donor vector, RP-Fib, contains a modified fiber gene, which is also floxed. Several variations of the smaller donor include a unique BspEI site in the HI loop for the incorporation of targeting ligands and/or a mutation in the receptor binding region of fiber. Additionally, the donor contains many convenient restriction enzyme recognition sites so genes other than fiber can be efficiently shuttled into pFex. The numerous shuttle vectors are described in detail below.
Using the described system, the fiber gene can be transferred into pFex either before (FIG. 4) or after (FIG. 5) the recombination with the E1 shuttle vector. Two separate fiber shuttle scaffolds have been constructed for either transfer stage. RP-Fib, which is kanamycin resistant, is applied for recombination prior to the E1 shuttle recombination (FIG. 4), and RPuc-Fib, which is ampicillin resistant, is applied for recombination after the E1 shuttle recombination. To increase the efficiency of the E1 shuttle recombination, pFex stable E. coli called bFex, can be used to overcome limitations in large plasmid transformation efficiency. This option is available for any pFex vector, after fiber exchange, if multiple E1 variations are needed. Both recombination pathways result in the same product, which can then be linearized with Pac I digestion, and transfected into a mammalian cell packaging cell line, such as 293-HEK, for the creation of virus. A third fiber shuttle, RP-Blast-Fib, has been designed to allow for blasticidin selection at either stage of recombination.
B. Design and Methods for Producing pFex Components.
pFex was assembled through several steps. First, a segment called ‘distal to fiber Age I’ was created by PCR amplification of the adenovirus serotype 5 genome with primers AdE-Dist 5′ and AdE-Dist 3′ (Table 1). This product was then cloned into the TOPO-TA vector pCR-2.1, using TA cloning, to produce the vector Step 1 pFex (FIG. 6).
Second, a segment called ‘proximal to fiber’ was created by PCR amplification of the adenovirus serotype 5 genome with primers loxmve1 and loxmve2 (Table 1). This product was then cloned into Step 1 pFex using the Spe I and Age I restriction sites. The resulting vector is Step 2 pFex (FIG. 7).
The SacB gene was isolated from the vector pAJ200 using the Bgl II and Pvu I restriction sites. Next, the two half mutant lox sites, lox m2/66 and lox 71, were added by ligation with self annealed linkers 5′ lox m2/66 and 3′ lox m2/66, and 5′ lox 71 and 3′ lox 71, respectively (Table 1). The resulting floxed SacB gene was then subcloned into Step 2 pFex to create Step 3 pFex (FIG. 8).
Finally, the modified AdEasy segment containing SacB in place of fiber was removed with a double digest of SpeI and PacI. This product was then exchanged for the pre-existing region of fiber in pAdEasy-1. The final vector construct is called pFEX (FIG. 9). The final product was verified by sequencing using primers pFEX for 01-11 and pFEXrev01-11 (Table 1). Finally, a second version of pFEX, termed pFEX-p*, contains a mutation in the integrin binding domain of the penton gene, where RGD is mutated to RGE (FIG. 9).
The fiber shuttle vectors were also constructed in a stepwise manner. An existing adenovirus serotype 5 fiber vector, pBK-CMV-Fiber, was first digested with the restriction enzymes Spe I and Xho I. The linkers S-lox m2/71-X5 and S-lox m2/71-X5 (Table 2) were self annealed and then inserted into the vector at these sites, creating Step 1 Fiber Shuttle Lox m2/71 (FIG. 10). This product was then digested with restriction enzymes Acc65 I and Not I, and the linkers N-Lox 66-A-5 and N-Lox 66-A-3 (Table 2), were then ligated into this site. The final product was named RP-Fib (FIG. 11). Finally, the tripartite leader splice acceptor site was inserted downstream of the lox m2/71 site by annealing the primers splce1 (TCGAGAACTATCTTCATGTTGTTGCAGATGAAGCGCGCAAGACCGTCTGAAGATACCTTCAACCCCGTGTATC CATATGACACGGAAA) (SEQ ID NO.7) and splce 2 (CCGGTTTCCGTGTCATATGGATACACGGGGTTGAAGGTATCTTCAGACGGTCTTGCGCGCTTCATCTGCAACAACATGAAG ATAGTTC) (SEQ ID NO.8) and cloning this into XhoI/AgeI sites of all fiber shuttle vectors. All of the described RP-Fib vectors have a mutated fiber gene that contains a unique BspEI site in the gene's HI loop for the incorporation of targeting peptide sequences (FIG. 11). Additionally, some vectors have a mutated fiber gene were the coding region for T489AYT492, a known Coxsackie and Adenovirus Receptor (CAR) binding site, has been deleted (Roelvink, P. W. et al. (1999) Science, 286: 1568-1571).
The current fiber shuttle vectors are summarized in Table 3. All RP-Fib vectors contain genes encoding kanamycin resistance. A separate set of vectors, RPuc-Fib, contain the same floxed fiber genes; however, the vector base is pUC-19, which is amplicillin resistant (FIG. 12). These two separate selection antibiotics allow for fiber gene exchange to occur at multiple steps (FIGS. 4 & 5).
C. Recombination of pFex with E1 Shuttle Vectors
The pFex vector was recombined with two E1 region shuttle vectors, pAdTrack (FIG. 13) and pAdTrack-CMV-Luc (FIG. 14) to demonstrate working recombination in these regions. This recombination step is based on the previously described AdEasy system (He, T. C. et al. (I 998) Proc Natl Acad Sci USA, 95: 2509-2514).
To increase the chances of recombination, the RecA positive bacterial line BJ5183 was first stably transfected with pFex. This technique has been shown to significantly increase the number of recombinants with the AdEasy vectors (Zeng, M. et al. (2001) Biotechniques, 31: 260-262). Each pAdTrack vector was then transformed into pFex stable BJ5183 cells, followed by selection on 50 μg/ml Kanamycin. There are two desired recombination products that replace the Ampicillin resistance cassette of pFex, one where recombination takes place between the homologous adenoviral left and right hand regions, or a second where the homologous replication origins and adenoviral right hand regions recombine. Either product is acceptable as Pac I digestion produces the same desired adenoviral genome product. Here, all products were the result of recombination between the origins of replication and the adenoviral right hand region (FIG. 15). Recombination between the adenoviral left and right hand portions would have produced a Pac I digestion product 1.7 Kb smaller without an additional Nde I site. Later whole viral genome products demonstrate four bands following Nde I digestion, indicating the recombination between the origins of replication and adenoviral right hand regions (FIG. 16).
D. Recombination of pFex with Fiber Shuttle Vectors
The pFex vector was then recombined with the kanamycin resistant shuttle vectors Rp-Fib-1, Rp-Fib-2, Rp-Fib-3, and Rp-Fib-4 to demonstrate working Cre lox recombination. This fiber exchange reaction was facilitated by the Cre expressing bacteria, 294cre. For each shuttle vector, pFex and molar excess of the Fiber shuttle were co-transformed into 294cre cells by electroporation. These cells were then heat shocked for 20 minutes at 42° C. to induce Cre expression, and then incubated at 37° C., while shaking, for 2 hours to continue Cre expression and Cre based recombination. The formation of expected recombinants could be demonstrated by PCR amplification of a product using primers within pFex and Fiber (FIG. 17). This reaction was also equally successful with 294cre cells stably transformed with pFex. Primer sets with pFex demonstrate the presence of pFex in all samples. Here, an Mfe I+Rsr II fragment of the fiber shuttle, which still retains the floxed Fiber gene, was unable to recombine with pFex; although, it was later found that this fragment could produce recombinants, but with less efficiency than intact shuttle plasmid. There were no recombination specific products in a control reaction containing pFex and a CMV-Luc vector.
Each transformation was then selected on LB plates with 100 μg/ml ampicillin and 7% sucrose. A small number of colonies were found for each pFex recombination with Rp-Fib shuttle plasmids, indicating that approximately 1% of the pFex plasmids successfully recombined with Rp-Fib shuttles. It has been determined that the recombination products must be further transformed into a more stable, Cre recombinase negative bacterial line, such as DH5α, to isolate the desired products. We found 24/24 ampicillin and sucrose resistant DH5α colonies to contain the desired recombinants without any contaminating aberrant recombination products (FIG. 18). We have successfully recombined all four Rp-Fib shuttle vectors with pFex.
E. Recombination of pAdTrack-Fex and pAdTrack-Luc Fex with RPuc-Fib Shuttles
To demonstrate that the pFex vector can be recombined in both the E1 and Fiber region, the pAdTrack recombination products pAdTrack-Fex and pAdTrack-Luc Fex were recombined with all four RPuc-Fib shuttle vectors. As before, the larger pAdTrack-Fex vectors were co-transformed into 294cre cells with molar excess RPuc-Fib shuttle vectors. Cre expression was induced by heat shock at 42° C. for 20 minutes, followed by 2 hour incubation at 37° C. with shaking at 225 rpm. Recombination efficiency was assessed by selection on a variety of antibiotics, with and without sucrose selection (Table 4). These results indicate that approximately 0.5-7% of the large pAdTrack-Fex and pAdTrack-Luc-Fex vectors recombined successfully with the RPuc-Fib shuttle vectors (Table 5). This efficiency will be significantly improved with further optimization of the Cre recombination reaction and sucrose selection. One colony from each kanamycin and sucrose selection plate were amplified, the DNA isolated and then transformed into the more stable DH5α cell lines, followed by a final colony selection on kanamycin and sucrose. Xho I digestion of these products reveals that all are the result of fiber exchange, giving the desired 3.6 Kb product (FIG. 19). Further, sequencing confirmed both that Cre lox recombination occurred as predicted, and that the expected Fiber modification was incorporated into the viral genome for each shuttle vector.
F. Generation of Adenovirus
Adenovirus containing wild type fiber was generated with pFex for the purpose of directly comparing AdEasy and pFex derived virus. The E1 shuttle vector, pAdTrack, was recombined into the E1 region of both AdEasy-1 and pFex. The resulting pAdTrack-Fex vector was then recombined with a fiber shuttle encoding the Wild Type Fiber, Rpuc-WTFib (FIG. 20). The resulting pFex-based viral genome was termed “pAdTrack-WTFib”. Both pAdTrack-WTFib and pAdTrack-AdEasy viral plasmids were linearized with PacI and separately transfected into 293 cells for viral production. Both plasmids generated viable virus. These were concurrently amplified, harvested, and titered. The resulting viral titers were identical between AdTrack-AdEasy and AdTrack-WTfib virus (Table 6). Further, both virus were applied to 293 cells at low multiplicity of infection (MOI) and plaques size was compared by fluorescent microscopy (GFP) to determine if there were any pFex-related deleterious effects on viral replication. Both virus had identical plaque size (FIG. 21). Therefore, there appear to be no deleterious effects of the lox sites on viral production or lifecycle.
G. Cre Mediated Fiber Exchange in Mammalian Cells
The Cre-recombinase mediate fiber exchange is not limited to E. coli or plasmids. Fiberless pFex viral vectors can be packaged into working virus through complementary cell lines that express wild type fiber protein (a process known as psuedotyping). These pseudotyped pFex viral vectors can then be used to infect Cre expressing mammalian cells (293cre57) that have been transfected with fiber donor vectors (FIG. 22). Following recombination (2-5 days), cell lysate and supernatant are harvested and used to infect a non-Cre expressing packaging line, such as 293, 911, or 911-S11. The efficiency of recombination is such that 0.01% wild type fiber shuttle, in the background of mutant fiber shuttle, can be detected. This efficiency is great enough to generate an adenoviral peptide display library.
This strategy was used to generate a CAR de-targeted adenovirus, AdTrack-Fib2. To achieve this, 293cre57 cells were simultaneously transfected with 3 μg RPuc-Fib2 (ΔTAYT) and infected with pseudotyped AdTrack-Fex virus at an MOI of 1. Five days post transfection-infection, cell and supernatant were harvested and freeze-thawed. This was used to infect 911-S11 cells, a packaging cell line which expresses an anti-fiber single chain antibody (scFv) for internalization of CAR de-targeted virus. The resulting virus was plaque purified, amplified, and titered in 911-S11 cells. FIG. 23 demonstrates the de-targeted viral production where equal particle numbers (1000 particles per cell) were applied to 293 cells or anti-fiber scFv 911-S11 cells. The lack of infection in 293 cells demonstrates CAR de-targeting, while the 911-S11 cell infection demonstrates viable fiber-containing virus. A control virus, AdTrack-WTFib, demonstrates equal infectivity of 293 and 911-S11.
H. Conclusions
The pFex system offers a unique and highly efficient means of creating fiber-modified or re-targeted adenoviral vectors. This system is fully compatible with the existing AdEasy gene vector system, which is currently applied in the majority of adenoviral vector laboratories. The system is very flexible, allowing Fiber gene transfer before or after E1 cassette exchange. Further, modified fiber gene can be shuttled into intact viral genomes in Cre recombinase expressing mammalian cell lines. This system is ideal for generating and screening modified fiber adenoviral vectors. There is a great need for re-targeted vectors on all levels of biological research, from gene transfer into a traditionally difficult to infect or transfect cell line to the development of systemically targeted therapeutic virus. pFex offers a simple and efficient means to create viral vectors to reach these goals.
Incorporation By Reference
The contents of all references, patents, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (61)

1. A method for generating a recombinant adenoviral vector encoding a desired product, comprising the step(s) of co-transforming a cell expressing an enzyme that mediates homologous recombination with
a) a linearized shuttle plasmid encoding a selectable marker, and
b) a transfer plasmid, wherein the transfer plasmid comprises a fiber gene, and wherein the fiber gene is modified to contain a unique restriction site in the HI loop;
thereby allowing recombination of the plasmids to generate the recombinant adenoviral vector.
2. The method of claim 1, wherein the enzyme that mediates homologous recombination is RecA.
3. The method of claim 1, wherein the transfer plasmid is constructed by co-transforming into a cell expressing Cre recombinase
a) a donor plasmid, wherein the donor plasmid encodes the fiber gene flanked by lox sites and a second selectable marker, and
b) an acceptor plasmid, wherein the acceptor plasmid encodes a negatively selectable marker flanked by lox sites and a first selectable marker, and wherein the acceptor plasmid does not encode the fiber gene;
thereby allowing for recombination of the fiber gene and the negatively selectable marker.
4. The method of claim 3, wherein the lox sites are incompatible.
5. The method of claim 3, wherein the lox sites are mutated to result in unidirectional recombination.
6. The method of claim 3, wherein the negatively selectable marker is SacB.
7. The method of claim 1, wherein the unique restriction site is a BspEl site.
8. The method of claim 3, wherein the donor plasmid lox sites are Lox m2/66 and Lox 71.
9. The method of claim 3, wherein the acceptor plasmid lox sites are Lox m2/71 and Lox 66.
10. The method of claim 3, wherein the acceptor plasmid contains a kanamycin selectable marker.
11. The method of claim 3, wherein the donor plasmid contains an ampicillin selectable marker.
12. The method of claim 1, wherein the method further comprises selecting recombinant adenoviral vectors using the selectable marker.
13. The method of claim 12, wherein the selectable marker is kanamycin.
14. The method of claim 1, wherein the cell is a bacterial cell.
15. The method of claim 14, wherein the bacterial cell is an E. coli cell.
16. The method of claim 3, wherein the cell is a bacterial cell.
17. The method of claim 16, wherein the bacterial cell is an E. coli cell.
18. The method of claim 3, wherein the cell is a mammalian cell.
19. The method of claim 1, wherein the shuttle plasmid comprises a resistance gene and a nucleic acid segment encoding the desired product.
20. The method of claim 19, wherein the product is selected from the group consisting of a polypeptide, polypeptides, or fragments thereof, a nucleic acid, an aptamer, an RNAi, an siRNA, and an shRNA.
21. The method of claim 20, wherein the desired product is a polypeptide.
22. The method of claim 21, wherein the desired polypeptide is a therapeutic polypeptide.
23. The method of claim 19, wherein the nucleic acid segment is under control of a promoter.
24. The method of claim 23, wherein the promoter is a tissue specific promoter.
25. The method of claim 19, wherein the transfer plasmid comprises a resistance gene, and wherein the resistance gene in the shuttle plasmid is not the same as the resistance gene in the transfer plasmid.
26. The method of claim 2, wherein the shuttle plasmid contains a unique restriction site located between RecA recombination sites.
27. The method of claim 26, wherein the unique restriction site in the shuttle plasmid is a Pme I site.
28. The method of claim 1, wherein the shuttle plasmid is linearized with Pme I.
29. The method of claim 1, wherein the shuttle plasmid further comprises homologous recombination sites.
30. The method of claim 29, wherein the homologous recombination sites are RecA homologous recombination sites.
31. The method of claim 30, wherein the RecA homologous recombination sites are Ad5 left and Ad5 right.
32. The method of claim 1, wherein the transfer plasmid further comprises homologous recombination sites.
33. The method of claim 32, wherein the homologous recombination sites are RecA homologous recombination sites.
34. The method of claim 33, wherein the RecA homologous recombination sites are Ad5 left and Ad5 right.
35. A method for generating a recombinant adenoviral vector encoding a desired gene product, the method comprising the step(s) of co-transforming a cell expressing Cre recombinase with
a) a donor plasmid, wherein the donor plasmid encodes a fiber gene flanked by lox sites, wherein the fiber gene is modified to contain a unique restriction site in the HI loop, and
b) a shuttle-acceptor plasmid encoding a negatively selectable marker, wherein the shuttle-acceptor plasmid does not encode the fiber gene;
thereby allowing for recombination of the fiber gene and the negatively selectable marker to generate the recombinant adenoviral vector.
36. The method of claim 35, wherein the shuttle-acceptor plasmid encoding the negatively selectable marker is constructed by co-transforming a cell expressing an enzyme that mediates homologous recombination with
a) an acceptor plasmid, wherein the acceptor plasmid encodes the negatively selectable marker flanked by lox sites and a first selectable marker, and
b) a linearized shuttle plasmid encoding a second selectable marker,
thereby allowing for recombination of the first selectable marker and the second selectable marker to generate the shuttle-acceptor plasmid encoding the negatively selectable marker.
37. The method of claim 36, wherein the enzyme that mediates homologous recombination is RecA.
38. The method of claim 35, wherein the lox sites are incompatible.
39. The method of claim 35, wherein the lox sites are mutated to result in unidirectional recombination.
40. The method of claim 35, wherein the negatively selectable marker is SacB.
41. The method of claim 35, wherein the unique restriction site is a BspEl site.
42. The method of claim 35, wherein the donor plasmid lox sites are Lox m2/66 and Lox 71.
43. The method of claim 36, wherein the acceptor plasmid lox sites are Lox m2/71 and Lox 66.
44. The method of claim 36, wherein the acceptor plasmid contains a kanamycin selectable marker.
45. The method of claim 35, wherein the donor plasmid contains an ampicillin selectable marker.
46. The method of claim 36, wherein the method further comprises selecting recombinant adenoviral vectors using the second selectable marker or the negatively selectable marker.
47. The method of claim 46, wherein the second selectable marker is kanamycin and the negatively selectable marker is SacB.
48. The method of claim 35, wherein the cell is a bacterial cell.
49. The method of claim 48, wherein the bacterial cell is an E. coli cell.
50. The method of claim 36, wherein the cell is a bacterial cell.
51. The method of claim 50, wherein the bacterial cell is an E. coli cell.
52. The method of claim 35, wherein the cell is a mammalian cell.
53. The method of claim 36, wherein the shuttle plasmid comprises a resistance gene and a nucleic acid segment encoding the desired product.
54. The method of claim 53, wherein the desired product is selected from the group consisting of a polypeptide, polypeptides, or fragments thereof, a nucleic acid, an aptamer, an RNAi, an siRNA, and an shRNA.
55. The method of claim 54, wherein the desired product is a polypeptide.
56. The method of claim 55, wherein the desired polypeptide is a therapeutic polypeptide.
57. The method of claim 53, wherein the nucleic acid segment is under control of a promoter.
58. The method of claim 57, wherein the promoter is a tissue specific promoter.
59. The method of claim 37, wherein the shuttle plasmid contains a unique restriction site located between RecA recombination sites.
60. The method of claim 59, wherein the unique restriction site in the shuttle plasmid is a Pme I site.
61. The method of claim 36, wherein the shuttle plasmid is linearized with Pme I.
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